Programming of cells for tolerogenic therapies

ABSTRACT

Biomaterial systems, e.g., gel scaffolds, are used in vivo to recruit immune cells and promote their activation towards a non-inflammatory phenotype, thereby leading suppression of inflammation. The compositions and methods are useful to reduce the severity of autoimmunity, chronic inflammation, allergy, and periodontal disease.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/386,950 filed Jan. 25, 2012, which is a national stage application,filed under 35 U.S.C. §371, of International Application No.PCT/US2010/044117 filed Aug. 2, 2010, which claims priority to U.S.Application No. 61/230,169 filed Jul. 31, 2009, the contents of whichare hereby incorporated by reference in their entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was funded in part by the U.S. Government under grantnumber 5R01DE019917-02 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to immune tolerance.

BACKGROUND

Aberrant or misregulated immune responses are underlying mechanisms ofnumerous pathological conditions. Such conditions include autoimmunedisorders and conditions characterized by chronic inflammation.

Autoimmunity is a condition where the immune system mistakenlyrecognizes host tissue or cells as foreign. Autoimmune diseases affectmillions of individuals worldwide. Common autoimmune disorders includetype 1 diabetes mellitus, Crohn's disease, rheumatoid arthritis, andmultiple sclerosis.

Chronic inflammation has been implicated in cancer, diabetes,depression, heart disease, stroke, Alzheimer's Disease, periodontitis,and many other pathologies. Aberrant or misregulated immune responsesare also implicated in asthma and allergy, e.g., asthma is a prevalentdisease with many allergen triggers.

SUMMARY

The invention provides a solution to the long standing clinical problemsof autoimmunity, allergy/asthma, and chronic or inappropriateinflammation in the body, e.g., inflammation that leads to tissue/organdamage and destruction. The compositions and methods direct the immuneresponse of an individual away from a pathological or life-threateningresponse and toward a productive or non-damaging response. Dendriticcells (DCs) play a major role in protecting against autoimmune disease.Regulatory T cells (Treg) also play an important part in inhibitingharmful immunopathological responses directed against self or foreignantigens. These cell types represent targets to manipulate for thepurpose of redirecting the immune response to provide a non-inflammatoryand non-destructive state.

Accordingly, the invention features a scaffold composition comprising anantigen, a recruitment composition, and a tolerogen. This scaffoldcomposition is useful for reduction of autoimmunity. The antigen is apurified composition (e.g., protein) or is a prepared cell lysate fromcells to which an undesired immune response is directed. Exemplaryrecruitment compositions include granulocyte-macrophage colonystimulating factor (GM-CSF; AAA52578), FMS-like tyrosine kinase 3 ligand(AAA17999.1), N-formyl peptides, fractalkine (P78423), or monocytechemotactic protein-1 (P13500.1). Exemplary tolerogens (i.e., agentsthat induce immune tolerance or a reduction in an immune response)include thymic stromal lymphopoietin (TSLP; Q969D9.1)), dexamethasone,vitamin D, retinoic acid, rapamycin, aspirin, transforming growth factorbeta (P01137), interleukin-10 (P01137), vasoactive intestinal peptide(CAI21764), or vascular endothelial growth factor (AAL27435). Thescaffold optionally further comprises a Th1 promoting agent such as atoll-like receptor (TLR) agonist, e.g., a polynucleotide such as CpG.Th1 promoting agents are often characterized by pathogen-associatedmolecular patterns (PAMPs) or microbe-associated molecular patterns(MAMPs) or alarmins PAMPs or MAMPs are molecules associated with groupsof pathogens, that are recognized by cells of the innate immune systemvia TLRs. For example, bacterial Lipopolysaccharide (LPS), an endotoxinfound on the gram negative bacterial cell membrane of a bacterium, isrecognized by TLR 4. Other PAMPs include bacterial flagellin,lipoteichoic acid from Gram positive bacteria, peptidoglycan, andnucleic acid variants normally associated with viruses, such asdouble-stranded RNA (dsRNA) or unmethylated CpG motifs. Thus, additionalexemplary Th1 promoting agents comprise a TLR 3, 4, or 7 agonist such aspoly (I:C), LPS/MPLA (monophosphate lipid A), or imiquimod,respectively. Exemplary TLR ligands include the following compounds:TLR7 Ligands (human & mouse TLR7)—CL264 (Adenine analog), Gardiquimod™(imidazoquinoline compound), Imiquimod (imidazoquinoline compound), andLoxoribine (guanosine analogue); TLR8Ligands (human TLR8 & mouseTLR7)—Single-stranded RNAs; E. coli RNA; TLR7/8 Ligands—(human, mouseTLR7 & human TLR8)—CL075 (thiazoloquinoline compound), CL097(water-soluble R848), imidazoquinoline compound, Poly(dT) (thymidinehomopolymer phosphorothioate oligonucleotide (ODN)), and R848(Imidazoquinoline compound).

The scaffolds mediate sustained release of the factors loaded therein ina controlled spatio-temporal manner. For example, the factors arereleased over a period of days (e.g., 1, 2, 3, 4, 5, 7, 10, 12, 14 daysor more) compared to bolus delivery of factors or antigens. Bolusdelivery often leads to little or no effect due to short-termpresentation in the body, adverse effects, or an undesirable immuneresponse if very high doses are provided, whereas scaffold deliveryavoids such events. Preferably, the scaffold is made from anon-inflammatory polymeric composition such as alginate, poly(ethyleneglycol), hyaluronic acid, collagen, gelatin, poly (vinyl alcohol),fibrin, poly (glutamic acid), peptide amphiphiles, silk, fibronectin,chitin, poly(methyl methacrylate), poly(ethylene terephthalate),poly(dimethylsiloxane), poly(tetrafluoroethylene), polyethylene,polyurethane, poly(glycolic acid), poly(lactic acid),poly(caprolactone), poly(lactide-co-glycolide), polydioxanone,polyglyconate, BAK; poly(ortho ester I), poly(ortho ester) II,poly(ortho ester) III, poly(ortho ester) IV, polypropylene fumarate,poly[(carboxy phenoxy)propane-sebacic acid],poly[pyromellitylimidoalanine-co-1,6-bis(p-carboxy phenoxy)hexane],polyphosphazene, starch, cellulose, albumin, polyhydroxyalkanoates, orothers known in the art (Polymers as Biomaterials for Tissue Engineeringand Controlled Drug Delivery. Lakshmi S, Nair & Cato T. Laurencin, AdvBiochem Engin/Biotechnol (2006) 102: 47-90 DOI 10.1007/b137240).Alternatively, a polymeric composition that provides a low level ofinflammation may also be useful, as it may aid in recruitment and/oractivation of dendritic cells, particularly biasing the cells towards aTh1 response. Poly(lactide), poly(glycolide), their copolymers, andvarious other medical polymers may also be useful in this regard.Ceramic or metallic materials may also be utilized to present thesefactors in a controllable manner. For example, calcium phosphatematerials are useful. In the context of bone, silica or other ceramicsare also be useful.

In some examples, composite materials may be utilized. For example,immune activating factors (e.g., antigen, tolerogen, or Th1 promotingagent) are encapsulated in microspheres such as poly(lactide-co-glycolide) (PLG) microspheres, which are then dispersed in ahydrogel such as an alginate gel. Cells, e.g., DCs and/or Tregs, arerecruited to or near the surface, or into the scaffold, where they mayreside for some period of time as they, are exposed to antigens andother factors described above, and then migrate away to bodily tissuessuch as lymph nodes, where they function to induce immune tolerance.Alternatively, the scaffold with cells may create a mimic of a secondarylymphoid organ. Following contact with the loaded scaffolds, such cellsbecome activated to redirect the immune response from a Th1/Th2/Th17response (autoimmunity and chronic inflammation) to a Treg response orfrom a pathogenic Th2 state toward a Th1 state (in the case ofallergy/asthma). Directing the immune response away from a Th2 responseand toward a Treg response leads to a clinical benefit in allergy,asthma. For autoimmunity, the therapeutic method is carried out byidentifying a subject suffering from or at risk of developing anautoimmune disease and administering to the subject the loaded scaffolds(antigen (autoantigen)+recruitment composition+tolerogen), leading to analteration in the immune response from a Th1/Th17 to T regulatory biasedimmune response. For allergy/asthma, the therapeutic method is carriedout by identifying a subject suffering from or at risk of developing anallergic response or asthma and administering to the subject the loadedscaffolds (antigen (allergen)+recruitment composition+adjuvant(Th1-promoting adjuvant)), thereby leading to an alteration in theimmune response from a Th2 response to a Th1 biased immune response(allergy/asthma).

A method of preferentially directing a Th1-mediated antigen-specificimmune response is therefore carried out by administering to a subject ascaffold comprising an antigen, a recruitment composition and anadjuvant. A dendritic cell is recruited to the scaffold, exposed toantigen, and then migrates away from the scaffold into a tissue of thesubject, having been educated/activated to preferentially generate a Th1immune response compared to a pathogenic Th2 immune response based onthe exposure. As a result, the immune response is effectively skewed orbiased toward the Th1 pathway versus the Th2 pathway. Such a bias isdetected by measuring the amount and level of cytokines locally or in abodily fluid such as blood or serum from the subject. For example, a Th1response is characterized by an increase in interferon-γ (IFN-gamma). Asdiscussed above, the scaffold optionally also comprises a Th1 promotingagent.

The compositions and methods are suitable for treatment of humansubjects; however, the compositions and methods are also applicable tocompanion animals such as dogs and cats as well as livestock such ascows, horses, sheep, goats, pigs.

The scaffolds are useful to manipulate the immune system of anindividual to treat a number of pathological conditions that arecharacterized by an aberrant, misdirected, or otherwise inappropriateimmune response, e.g., one that causes tissue damage or destruction.Such conditions include autoimmune diseases. For example, a method ofreducing the severity of an autoimmune disorder is carried out byidentifying a subject suffering from an autoimmune disorder andadministering to the subject a scaffold composition comprising anantigen (e.g., a purified antigen or a processed cell lysate), arecruitment composition, and a tolerogen. Preferably, the antigen isderived from or associated with a cell to which a pathologic autoimmuneresponse is directed. In one example, the autoimmune disorder is type 1diabetes and the antigen comprises a pancreatic cell-associated peptideor protein antigen, e.g., insulin, proinsulin, glutamic aciddecarboxylase-65 (GAD65), insulinoma-associated protein 2, heat shockprotein 60, ZnT8, and islet-specific glucose-6-phosphatase catalyticsubunit related protein or others as described in Anderson et al.,Annual Review of Immunology, 2005. 23: p. 447-485; or Waldron-Lynch etal., Endocrinology and Metabolism Clinics of North America, 2009. 38(2):p. 303). In another example, the autoimmune disorder is multiplesclerosis and the peptide or protein antigen comprises myelin basicprotein, myelin proteolipid protein, myelin-associated oligodendrocytebasic protein, and/or myelin oligodendrocyte glycoprotein. Additionalexamples of autoimmune diseases/conditions include Crohn's disease,rheumatoid arthritis, Systemic lupus erythematosus, Scleroderma,Alopecia areata, Antiphospholipid antibody syndrome, Autoimmunehepatitis, Celiac disease, Graves' disease, Guillain-Barre syndrome,Hashimoto's disease, Hemolytic anemia, Idiopathic thrombocytopenicpurpura, inflammatory bowel disease, ulcerative colitis, inflammatorymyopathies, Polymyositis, Myasthenia gravis, Primary biliary cirrhosis,Psoriasis, Sjögren's syndrome, Vitiligo, gout, celiac disease, atopicdermatitis, acne vulgaris, autoimmune hepatitis, and autoimmunepancreatitis.

The scaffolds are also useful to treat or reduce the severity of otherimmune disorders such as a chronic inflammatory disorder orallergy/asthma. In this context, the method includes the steps ofidentifying a subject suffering from chronic inflammation orallergy/asthma and administering to the subject a scaffold compositioncomprising an antigen associated with that disorder, a recruitmentcomposition, and an adjuvant. The vaccine is useful to reduce acuteasthmic exacerbations or attacks by reducing/eliminating the pathogenicresponse to the allergies. In the case of allergy and asthma, theantigen comprises an allergen that provokes allergic symptoms, e.g.,histamine release or anaphylaxis, in the subject or triggers an acuteasthmatic attack. For example, the allergen comprises (Amb a 1 (ragweedallergen), Der p2 (Dermatophagoides pteronyssinus allergen, the mainspecies of house dust mite and a major inducer of asthma), Betv 1 (majorWhite Birch (Betula verrucosa) pollen antigen), Aln g I from Alnusglutinosa (alder), Api G I from Apium graveolens (celery), Car b I fromCarpinus betulus (European hornbeam), Cor a I from Corylus avellana(European hazel), Mal d I from Malus domestica (apple), phospholipase A2(bee venom), hyaluronidase (bee venom), allergen C (bee venom), Api m 6(bee venom), Fel d 1 (cat), Fel d 4 (cat), Gal d 1 (egg), ovotransferrin(egg), lysozyme (egg), ovalbumin (egg), casein (milk) and whey proteins(alpha-lactalbumin and beta-lactaglobulin, milk), and Ara h 1 throughAra h 8 (peanut). The compositions and methods are useful to reduce theseverity of and treat numerous allergic conditions, e.g., latex allergy;allergy to ragweed, grass, tree pollen, and house dust mite; foodallergy such as allergies to milk, eggs, peanuts, tree nuts (e.g.,walnuts, almonds, cashews, pistachios, pecans), wheat, soy, fish, andshellfish; hay fever; as well as allergies to companion animals,insects, e.g., bee venom/bee sting or mosquito sting. Preferably, theantigen is not a tumor antigen or tumor lysate.

Also within the invention are vaccines comprising the loaded scaffold(s)described above and a pharmaceutically-acceptable excipient forinjection or implantation into a subject for the to elicit antigenspecific immune tolerance to reduce the severity of disease. Otherroutes of administration include topically affixing a skin patchcomprising the scaffold or delivering scaffold compositins by aerosolinto the lungs or nasal passages of an individual.

In addition to the conditions described above, the scaffolds and systemsare useful for treatment of periodontitis. One example of a biomaterialsystem for use in vivo that recruits dendritic cells and promotes theiractivation towards a non-inflammatory phenotype comprises a biomaterialmatrix or scaffold, e.g., a hydrogel such as alginate, and a bioactivefactor such as GM-CSF or thymic stromal lymphopoietin (TSLP) for use indental or periodontal conditions such as periodontitis. Periodontitis isa destructive disease that affects the supporting structures of theteeth including the periodontal ligament, cementum, and alveolar bone.Periodontitis represents a chronic, mixed infection by gram-negativebacteria, such as Porphyromonas gingivalis, Prevotella intermedia,Bacteroides forsythus, Actinobacillus actinomycetemcomitans, and grampositive organisms, such as Peptostreptococcus micros and Streptococcusintermedius.

The methods address regulatory T-cell modulation of inflammation inperiodontal disease. DCs can elicit anergy and apoptosis in effectorcells in addition to inducing regulatory T cells. Other mechanismsinclude altering the balance between Th1, Th2, Th17 and T regs. Forexample, TSLP is known to enhance Th2 immunity and in addition toincreasing T reg numbers could increase the Th2 response. The materialsrecruit and program large numbers of tolerogenic DCs to promoteregulatory T-cell differentiation and mediate inflammation in rodentmodels of periodontitis. More specifically, the recruitment, appropriateactivation, and migration to the lymph nodes of appropriately activatedDCs leads to the formation of high numbers of regulatory T-cells, anddecreased effector T-cells, reducing periodontal inflammation.

Another aspect of the present invention addresses the mediation ofinflammation in concert with promotion of regeneration. In particular,plasmid DNA (pDNA) encoding BMP-2, delivered from the material systemthat suppresses inflammation, reduces inflammation via DC targeting andenhances the effectiveness of inductive approaches to regeneratealveolar bone in rodent models of periodontitis. For example,significant alveolar bone regeneration results from a material thatfirst reduces inflammation, and then actively directs bone regenerationvia induction of local BMP-2 expression.

The invention provides materials that function to modulate theinflammation-driven progression of periodontal disease, and thenactively promote regeneration after successful suppression ofinflammation. Moreover, the compositions and methods described hereincan be translated readily into new materials for guided tissueregeneration (GTR). Unlike current GTR membranes that simply provide aphysical barrier to cell movement, the new materials actively regulateslocal immune and tissue rebuilding cell populations in situ. Morebroadly, inflammation is a component of many other clinical challengesin dentistry and medicine, including Sjogren's and other autoimmunediseases, and some forms of temporomandibular joint disorders. Thepresent invention has wide utility in treating many of these diseasescharacterized by inflammation-mediated tissue destruction. Further, thematerial systems also provide novel and useful tools for basic studiesprobing DC trafficking, activation, T-cell differentiation, and therelation between the immune system and inflammation. In addition to theconditions and diseases described above, the compositions and methodsare also useful in wound healing, e.g., to treat smoldering wounds,thereby altering the immune system toward healing and resolution of thewound.

Polypeptides and other compositions used to load the scaffolds arepurified or otherwise processed/altered from the state in which theynaturally occur. For example, a substantially pure polypeptide, factor,or variant thereof is preferably obtained by expression of a recombinantnucleic acid encoding the polypeptide or by chemically synthesizing theprotein. A polypeptide or protein is substantially pure when it isseparated from those contaminants which accompany it in its naturalstate (proteins and other naturally-occurring organic molecules).Typically, the polypeptide is substantially pure when it constitutes atleast 60%, by weight, of the protein in the preparation. Preferably, theprotein in the preparation is at least 75%, more preferably at least90%, and most preferably at least 99%, by weight. Purity is measured byany appropriate method, e.g., column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis. Accordingly, substantially purepolypeptides include recombinant polypeptides derived from a eucaryotebut produced in E. coli or another procaryote, or in a eucaryote otherthan that from which the polypeptide was originally derived.

In some situations, dendritic cells or other cells, e.g., immune cellssuch as macrophages, B cells, T cells, used in the methods are purifiedor isolated. With regard to cells, the term “isolated” means that thecell is substantially free of other cell types or cellular material withwhich it naturally occurs. For example, a sample of cells of aparticular tissue type or phenotype is “substantially pure” when it isat least 60% of the cell population. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99% or 100%, of the cell population. Purity is measured by anyappropriate standard method, for example, by fluorescence-activated cellsorting (FACS). In other situations, cells are processed, e.g.,disrupted/lysed and the lysate fractionated for use as an antigen in thescaffold

These and other capabilities of the invention, along with the inventionitself, will be more fully understood after a review of the followingfigures, detailed description, and claims. All references cited hereinare hereby incorporated by reference. Sequences are publically availableonline using Entrez protein data base atwww.ncbi.nlm.nih.gov/genbank/using the sequence identifiers providedherein.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the immune response role in periodontal disease(PD). The infection of PD typically leads to the formation of activateddendritic cells, which lead to generation of effector T-cells, andchronic inflammation in the tissue that over time results in boneresorption.

FIG. 2 is a schematic of an approach to ameliorate PD inflammation andpromote bone regeneration in an embodiment of the present invention. Thegel delivered into the site of inflammation first releases GM-CSF andTSLP, to promote formation of tolerant DCs (tDCs) from immature DCs, andblock DC activation. The increased ratio of tolerant DCs/activated DCspromotes formation of regulatory T-cells (Tregs), and inhibit effectorT-cells. This reduces process inflammation and accompanying boneresorption, and instead promotes resolution of inflammation. The gelreleases pDNA encoding for BMP-2 as inflammation subsides, and localBMP-2 expression drives bone regeneration. Bracket A addresses therelation between gel-delivery of GM-CSF and TSLP and subsequentgeneration of tDCs. Bracket B shows the resultant impact on formation ofTregs and inflammation, and bracket C shows on-demand pDNA delivery fromgels and the impact on bone regeneration following amelioration ofinflammation.

FIGS. 3A-D show data related to the concentration dependent effects ofGM-CSF on DC proliferation, recruitment, activation and emigration invitro. FIG. 3A shows the in vitro recruitment of JAWSII DCs induced bythe indicated concentrations of GM-CSF in transwell systems. Migrationcounts measured at 12 hours. FIG. 3B is the effects of GM-CSFconcentration on the proliferation of JAWSII DCs. 0 (white bar), 50(grey bar), and 500 ng/ml (black bar) of GM-CSF. FIG. 3C shows theeffects of the indicated concentrations of GM-CSF on JAWS II DCemigration from the top wells of transwell systems toward mediasupplemented with 300 ng/ml CCL19. Migration counts taken at 6 hours.FIG. 3D are representative photomicrographs of TNF-α and LPS stimulatedJAWSII DCs cultured in 5-50 or 500 ng/ml GM-CSF and stained for theactivation markers MHCII and CCR7. Scale bar in FIG. 3D—20 μm. Values inFIGS. 3A-C represent mean and standard deviation (n=4); *P<0.05;**P<0.01

FIGS. 4A-F present data on the in vivo control of DC recruitment andprogramming. FIG. 4A is the release profile of GM-CSF from polymers thatdemonstrates a large initial burst, to create high early concentrationsof GM-CSF in tissue. FIG. 4B shows H&E staining of tissue sectionsfollowing explanation from subcutaneous pockets in the backs of C57BL/6Jmice after 14 days: Blank polymers, and GM-CSF (3000 ng) loadedpolymers. FIG. 4C shows FACS plots of cells isolated from explantedpolymers after 28 days and stained for the DC markers, CD11c and CD86implanted. Numbers in FACS plots indicate the percentage of the cellpopulation positive for both markers. FIG. 4D is the percentage of totalcells that were positive for the DC markers CD11c and CD86, in blank(--∘--) and GM-CSF (-•-) loaded polymers as a function of time postimplantation. FIG. 4E shows the total number of DCs isolated from blank(--∘--) and GM-CSF (-•-) loaded polymers as a function of time postimplantation. FIG. 4F shows the fractional increase in CD11c(+)CD86(+)DCs isolated from polymers at day-14 after implantation in response todoses of 1000, 3000 and 7000 ng of GM-CSF as compared to the control.Scale bar—500 μm. Values in FIGS. 4A, 4D, 4E, and 4F represent mean andstandard deviation (n=4 or 5); *P<0.05; **P<0.01.

FIGS. 5 (Table 1) and 5A-E reflect the potency of a material system thatdelivers TSLP and GM-CSF to PD lesion in induction of tolerogenic DC.FIGS. 5A-5C shows cytokine production by CD11+DC induced in vitro frombone marrow cells with GM-CSF in the presence or absence of TSLP, VIP,or TGF-β (7 day incubation). The in vitro incubation of mononuclearcells isolated from the bone marrow (BM) of C57BL/6 mice with GM-CSF andTSLP (100 ng/ml, respectively) for 7 days up-regulated thedifferentiation of tolerogenic DC that produced high IL-10 (FIG. 5A) andlow IL-6 (FIG. 5B) and IL-12 (FIG. 5C). While TGF-β (100 ng/ml) alsoshowed a similar trend to TSLP in the induction of tolerogenic DC, VIPdid not up-regulate the ability of DCs to produce IL-10. The surfacephenotypes of CD11c+DC in the BM culture were monitored by flowcytometry and the proportionality of each phenotype is expressed as apercent (%) of the total mononuclear cells (MNC) (FIG. 5, Table 1). Thedouble-color confocal microscopy showed that the gingival injection ofgel (1.5 μl) with GM-SCF (1 μg) and TSLP (1 μg) increased CD11c+cellswhich produce IL-10 in the mouse periodontal bone loss lesion (FIG. 5E;7 days after injection), compared to the control bone loss lesion whichdid not received injection (FIG. 5D).

FIGS. 6A-B demonstrate control over local T-cell numbers, andantigen-specific CD8 T-cells. FIG. 6A shows FACS histograms of CD8(+)cell tissue infiltration with blank vehicle (gray line), vehicle loadedwith 3000 ng GM-CSF and 100 μg CpG-ODN alone (dashed line), and vehicleloaded with GM-CSF and antigens (black line). FIG. 6B showscharacterization of TRP2-specific CD8 T-cells. Splenocytes from naïvemice (naïve) and mice receiving vehicles containing antigen+GM-CSF+CpGat day 30 (vaccinated) were stained with anti-CD8-FITC Ab, anti-TCR-APCAb, and Kb/TRP2 pentamers. The elliptical gates in the upper rightquadrant represent the TRP2-specific, CD8(+) T cells and numbers providepercentage of positive cells. Values represent the mean.

FIG. 7 shows vertical bone loss induced in a mouse model of PD. 7A is animage of a human clinical case of vertical periodontal bone loss(picture taken at the flap operation). 7B shows GTR-membrane appliedonto the vertical bone loss. 7 a-7 f are anatomical demonstration ofvertical bone loss induced in the mouse model of periodontitis. Thirtydays following PPAIR-induction in the mice harboring oral Pp by systemicimmunization (s.c.) with fixed Aa, animals were sacrificed anddefleshed. 7 a and 7 b: control mice which did not receive immunizationwith fixed Aa; 7 c-7 e: mice developed vertical periodontal bone lossaround the maxillary molars by systemic immunization with fixed Aa; 7 g:histochemical (HE-staining) image of decalcified tissue section ofcontrol periodontally healthy mouse; 7 h: histochemical (HE-staining)image of mouse which developed PD accompanied by vertical periodontalbone loss (higher magnification image clearly demonstrates extensiveneutrophil infiltration).

FIGS. 8A-F demonstrate adoptive transfer of ex vivo-expanded Treg toPp-harboring mice abrogated periodontal bone resorption induced byPPAIR. Following the protocol reported by Zheng et al., these resultshow ex vivo expansion of FOXP3+CD25+T cells by culture of spleen cellsisolated from Aa-immunized mice (i.p. injection of Aa 10¹⁰/mouse) in thepresence of recombinant human TGFb1 (Peprotech), mouse IL-2 (Peprotech),and fixed Aa, as antigens. After ex vivo stimulation for 3 days, thepercentage of FOXP3+CD25+Treg cells in the total lymphocytes increasedfrom 5.5% on day-0 to 15.0% on day-3 (FIG. 8A, upper 2 graphs).Similarly, the percentage of FOXP3+CD4+Treg cells also increased in theculture (FIG. 8A, lower 2 graphs). After 6 days of ex vivo stimulation,the percentage of FOXP3+CD25+ cells reached 23.3% of the totallymphocytes and 79.8% of the total CD4 T cells. The CD4+ cells wereisolated by the magnet beads-based negative selection technique(TGF/IL-2/Aa/CD4+T cells). TGF/IL-2/Aa/CD4+Treg cells were labeled withCFSE (5 μM, in PBS, 8 min, MolecularProbe) and adoptively transferred(10⁶/mouse). The localization of CFSE-labeled cells was confirmed byflow cytometry in gingival tissue and cervical lymph nodes (not shown).The TGF/IL-2/Aa/CD4+Treg cells (2×10⁴/well) were treated with MitomycinC (MMC) and co-cultured with Aa-specific Th1 effector cells (2×10⁴/well)in the presence of MMC-treated spleen APC (2×105/well) and Aa antigens.CD25+ cells in original spleen CD4+T cells were depleted by cytotoxicanti-CD25 monoclonal antibody (PC61, rat IgG2a, Pharmingen) in thepresence of mouse complement sera (Sigma). Such CD25-depleted spleenCD4+T cells were also included after adjusting the cell number.Proliferation of Th1 effector cells was monitored by 3H-thymidine assay(4 days), and sRANKL concentration in the culture supernatant wasmeasured by ELISA (FIG. 8B). The TGF/IL-2/Aa/CD4+ cells were alsoadoptively transferred into Pp-harboring mice, and bone resorption (FIG.8C), concentration of IFN-g (FIG. 8D), sRANKL (FIG. 8E) and IL-10 (FIG.8F) in the gingival tissue homogenates were all measured on Day-30. *,Significantly different from control by Student's t test (P<0.05). **,Significantly different from the Aa (s.c.) injection alone (*) byStudent's t test (P<0.05).

FIGS. 9A-O show expansion of FOXP3+T cells in mouse gingival tissue andlocal lymph nodes (LN) by GM-CSF/TSLP delivery polymer. FOXP3-EGFP-KImice which previously developed periodontal bone-resorption-socket(maxillary molars) by PPAIR-mediated PD induction received a gingivalinjection of a total 1.5 μl of (1) control empty polymer, (2) polymerwith GM-CSF (1 μg), and (3) polymer with GM-CSF (1 μg)+TSLP (1 μg). Thelocal cervical lymph nodes (CLN) and maxillary jaws were removed fromthe sacrificed animals at Day-7 after the injection of polymer. EGFP+cells (=FOXP3+Treg cells) in the CLN were monitored by flow cytometry(FIGS. 9A, 9B and 9C). The presence of FOXP3+Treg cells in the mouseperiodontal bone loss lesion was evaluated using a fluorescent confocalmicroscope (FIGS. 9D-9K). FIG. 9D shows an illustration indicating theanatomical objects (tooth root, alveolar bone and inflammatoryconnective tissue), FIG. 9H shows a histochemical image (HE-staining) ofperiodontal bone loss lesion, FIGS. 9E-G show bright field images, FIGS.9I-K show fluorescent images. FIGS. 9E, 9H, and 9I show adjacent sectionof a mouse which did not receive polymer injection, FIGS. 9F and 9J showa mouse receiving polymer injection with GM-CSF, FIGS. 9G and 9K show amouse receiving polymer injection with GM-CSF+TSLP. Mouse gingivaltissue in the bone loss lesion that received GM-CSF/TSLP deliverypolymer showed CD11c+ cells and IL-10 around the FOXP3+T cellsinfiltrating in the foci (FIGS. 9N, 9O), whereas the control bone losslesion did not receive polymer injection showed little or no CD11C+cells or IL-10 in the tissue where the infiltrate of FOXP3 cells wasalso low (FIGS. 9L, 9M).

FIGS. 10A-D demonstrate that polymeric delivery of PEI-condensed pDNAencoding BMP leads to bone regeneration. Implantation of scaffolds ledto (FIG. 10A) long-term (15 week) expression of human BMP-4 in mice(immunohistochemistry; arrows indicate positive cells), and (FIG. 10B)significant regeneration of bone in critical size cranial defects, ascompared to blank polymers. Circles denote original area of bone defect,bone within the circle represents newly regenerated bone tissue.Statistically significant increases in the defect area filled withosteoid (FIG. 10C) and mineralized tissue (FIG. 10D), were found withcondensed pDNA delivery, as compared to blank polymers, or polymersloaded with an equivalent quantity of non-condensed pDNA. All data at 15weeks, and values represent mean and standard deviation. The datademonstrate control over the timing of pDNA release from alginate gelsvia control over gel degradation rate.

FIGS. 11A-B are line graphs demonstrating precise control over thetiming of pDNA release from alginate gels with ultrasound. Alginate gelsencapsulating pDNA were incubated in tissue culture medium, and anultrasound transducer was placed in the medium. Irradition (1 W) wasapplied to gels for 15 min daily; the release rate of pDNA was analyzedby collecting medium and quantifying pDNA in the solution. The baserelease rate of pDNA was minimal from the high molecular weight, slowlydegrading gels used in these studies.

FIG. 12 is a line graph showing pDNA release rate.

FIG. 13 is a schematic of an in vitro Treg development assay.

FIG. 14A is a diagram showing an overhead view of a petri dish, lightshading represents the collagen and DCs while the darker shading (innercircle) represents the alginate gel).

FIGS. 14B-C are dot plots showing bone marrow-derived dendritic cellchemokinesis in vitro to alginate containing hydrogels with or withoutGM-CSF. FIG. 14B (no GM-CSF); FIG. 14C (GM-CSF mixed in with alginate).

FIG. 14D is a list of average migration speed of dendritic cells in thepresence of GM-CSF and in the absence of GM-CSF (control).

FIG. 15 is a photograph of alginate gel scaffold material under the skinof a mouse. Scale bar is 5 mm.

FIGS. 16A-B are a series of photomicrographs showing recruitment of DCsto GM-CSF loaded alginate gels in vivo. FIG. 16A shows alginate gelswithout GM-CSF, and

FIG. 16B shows alginate gels containing GM-CSF.

FIG. 16C is a bar graph showing a quantification of cells in blank(alginate without GM-CSF) and GM-CSF loaded alginate gels.

FIG. 17 is a series of photomicrographs showing expression of Forkheadbox P3 (FoxP3) in cells adjacent to alginate gels releasing GM-CSF andThymic stromal lymphopoietin (TSLP) in vivo. Gels containing 3 μg ofGM-CSF and 0 μg (A, left panel) or 1 μg (B, right panel) of TSLP wereexplanted 7 days after injection. White dotted lines indicate the borderbetween the dermal tissue (left) and the alginate gels (right). Scalebars are 50 μm.

FIG. 18 is a line graph showing establishment of a murine type 1diabetes model.

FIG. 19 is a line graph showing quantification of euglycemic cellsfollowing administration of scaffolds containing PLGA-dex, ova, andGM-CSF; PLGA, ova, and GM-CSF, PLGA-dex, BSA and GM-CSF; and PLGA-dexand ova.

FIG. 20 is a bar graph showing ovalbumin-specific IgE in serum followingvaccination. The following vaccination groups were tested: no primaryvaccination; Ova scaffolds; Ova+GM-CSF scaffolds; Ova+GM-CSF+CpGscaffolds; and Bolus intraperitoneal (IP) injection ofOva+GM-CSF+CpG)/no scaffold. These data show that vaccination does notelicit pathogenic IgE antibodies.

FIG. 21 is a bar graph showing splenocyte interferon-γ (IFN-gamma)elaboration following ovalbumin administration.

FIG. 22 is a bar graph showing attenuation of anaphylactic shockfollowing vaccination with scaffolds containing CpG, GM-CSF, andovalbumin Temperature of test animals was measured following vaccinationand subsequent intraperitoneal challenge with ovalbumin.

DETAILED DESCRIPTION

The scaffolds and systems described herein mediate spatiotemporalpresentation of cues that locally control DC activation and bias theimmune response towards a non-pathogenic state. The scaffolds andmethods are used to treat subjects that have been identified assuffering from or at risk of developing diseases or disorderscharacterized by inappropriate immune activation. The biomaterialsystems (loaded scaffolds) recruit DCs and promote their activationtowards a tolerogenic or non-inflammatory phenotype(autoimmunity/inflammation) or an activated state (allergy/asthma) thatcorrects an aberrant or misregulated immune response that occurs in apathologic condition.

For autoimmune disease, the scaffolds comprise an antigen (autoantigen),a recruitment composition, and a tolerogen. For allergy or asthma, thescaffolds comprise and antigen (allergen), a recruitment composition,and an adjuvant (e.g., a Th1 promoting adjuvant such as CpG). Generationof Treg cells leads to clinical benefit by directing the immune responseaway from pathogenic T effectors and toward other immune effectors suchas Treg, Th1, Th17 arms of the immune system.

The vaccines attenuate diseases of pathogenic immunity by re-directingthe immune system from a Th1/Th17 to T regulatory biased immune response(autoimmunity) and a Th2 response to a Th1 biased immune response(allergy/asthma).

Scaffolds

Exemplary scaffolds were produced using PLG (for allergy or asthma) oralginate (for autoimmune diseases such as diabetes of forperiodontitis). PLG was compressed, gas foamed, and leached (porogens(that were later leached) 250 μm to 400 μm made up 90% of the compressedpowder) to create a porous material. Gels are typically 1-20% polymer,e.g., 1-5% or 1-2% alginate. Methods of making scaffolds are known inthe art, e.g., U.S. Ser. No. 11/638,796 or PCT/US2009/000914. Thepolymers are preferably crosslinked. For example, 1-2% alginate wascrosslinked ionically in the presence of a divalent cation (e.g.,calcium). Alternatively, to modify the spatiotemporal presentation ofmolecules and control degradation, the alginate is crosslinkedcovalently by derivatizing the alginate chains with molecules byoxidation with sodium periodate and crosslinking with adipicdihydrazide.

Vaccines that Attenuate Diseases of Pathogenic Immunity by Re-Directingthe Immune System from a Th1/Th17 to T Regulatory Biased Immune Response

GM-CSF enhanced chemokinesis of bone marrow dendritic cells in vitro.Alginate gels with or without GM-CSF (˜1 μg/gel) were placed in a petridish and surrounded with collagen containing bone marrow derived murinedendritic cells (FIG. 14A). The cells were followed for 8 hours usingtime-lapse imaging. The velocity of the cells was calculated frominitial and final position values and is plotted in FIGS. 14B and C inμm/min. Chemotaxis toward the alginate is given as the positive xcoordinate (positive x is directed radially inward). Each dot reflectsthe velocity of 1 cell, and each plot is representative of threeexperiments. The average migration speed of cells in the presence ofGM-CSF was 3.1 μm/min compared to 1.1 μm/min in the absence of GM-CSF.The speed of control and alginate gels is shown in FIG. 14D and wasfound to be significantly different at p<0.01. These data indicate thatGM-CSF increases the speed of movement of dendritic cells and thuspromotes dendritic cell migration.

To observe the biomaterial scaffold in vivo, alginate gels were injectedintradermally (FIG. 15). A 60 μL alginate gel was injected intradermallyinto the skin of a mouse. A photographic image was taken from the dermalside of the skin after euthanasia of the animal. Blue dye wasincorporated into alginate gels before crosslinking for visualization.

Recruitment of DCs to GM-CSF Loaded Alginate Gels In Vivo

FIGS. 16A-B show the results of immunofluorescent staining of sectionedskin containing alginate gels, showing nuclei, MHC class II, and CD11c.Gels containing 0 μg (A) or 3 μg (B) of GM-CSF were explanted 7 daysafter injection. White dotted lines indicate the border between thedermal tissue (left) and the alginate gels (right). Scale bars are 50μm. The area in tissue sections comprised of CD11c+ cells in blank gelsvs. gels loaded with 3 ug of GM-CSF was quantified after 7 days. Imageanalysis of stained sections was done using ImageJ (n=3animals/condition). *P<0.02. The data demonstrate that dendritic cellswere recruited to GM-CSF loaded gels in vivo.

T Regulatory (Treg) Cells are Recruited to GM-CSF/TSLP Loaded Gels

Treg cells were detected adjacent to alginate gels releasing GM-CSF andTSLP in vivo. TSLP promotes immune tolerance mediated by Treg cells andplays a direct and indirect role in regulating suppressive activities ofsuch cells. The main influence of TSLP peripherally is on the DCs;however, T cells have receptors for TSLP and are also affected. AlthoughTregs are instrumental as being the mode of therapeutic benefit forperiodontal disease, switch to a Th2 response (Th1→Treg/Th2) is alsoinvolved. For other diseases, a predominantly Treg response is desired;in the latter case, factors such as TGF-beta and IL-10 are utilized.

Cells were identified in FIG. 7 by detecting expression of FoxP3, atranscription factor specifically expressed in CD4+CD25+ Treg cells.Panels A and B of FIG. 17 show the results of immunofluorescent stainingof sectioned skin containing alginate gels, showing nuclei (grey dots)and FoxP3 (bright dots). All gels contained 3 μg of GM-CSF. The gel inpanel (A) did not contain TSLP (0 μg), whereas the gel in panel (B)contained 1 μg of TSLP. The gels were explanted 7 days after injectionand analyzed. White dotted lines indicate the border between the dermaltissue (left) and the alginate gels (right). Scale bars are 50 μm.Numerous bright dots (FoxP3-positive Treg cells) were detected usinggels containing both GM-CSF and TSLP. These data indicate that inincreased number of Treg cells are recruited to gels containing bothGM-CSF and TSLP compared to GM-CSF alone or alginate alone.

Dendritic Cell Immunotherapy for Type 1 Diabetes

The gel scaffolds described herein were evaluated in an art-recognizedautoimmune model for type 1 diabetes mellitus (T1DM). The model utilizesa transgenic animal that expresses ovalbumin (OVA) under the control ofthe rat insulin promoter (RIP) in the pancreas (RIP-OVA model). (see,e.g., Proc Natl Acad Sci USA. 1999 Oct. 26; 96(22): 12703-12707; orBlanas et al., 1996. Science 274(5293):1707-9.). OVA-specificCD8-positive (cytotoxic T) cells are adoptively transferredintravenously to induce and establish autoimmune diabetes. Morespecifically, the adoptively transferred T cells recognize the ovalbuminpresented on the pancreatic beta cells and attack these cells resultingin dampened insulin secretion and diabetes.

FIG. 18 shows percentages of euglycemic RIP-OVA mice over time followinginjection with various doses of OT-I splenocytes. 4 mice per group wereinjected with 6×10⁶, 2×10⁶, 0.67×10⁶, or 0.22×10⁶ activated CD8+Va2+OT-Isplenocytes administered i.v. Adoptive transfer of approximately 2×10⁶cells leads to diabetes in one week. Hyperglycemia was defined as 3consecutive days with a blood glucose reading above 300 mg/dL. Between0.67×10⁶ and 2×10⁶ T cells is a critical threshold for inducing disease.If cells are adminstered at this level concomitantly with therapies thatinfluence T cell fate as described herein, the number the number ofanimals that eventually become diabetic and the speed at which theybecome diabetic is substantially altered in comparison to controlanimals with the adoptive transfer of cells alone without therapy.

Using the same model system, alginate gel scaffolds were implantedintradermally. The percentage of euglycemic mice was then determinedover time following injection with 2×10⁶ OT-I splenocytes 10 days afteralginate intradermal implantation (FIG. 19). All animals received aninjection of alginate. Like TSLP, Dexamethasone (dex) is a compositionthat induces immune tolerance. In this experiment, dexamethasone wasencapsulated in poly (lactide-co-glycolide) (PLG) microspheres prior toloading into alginate gels to delay release of the dexamethasone. Thecomposition of the alginate gels was as follows: PLG: blank poly(lactide-co-glycolide) microspheres, PLGA-dex: dexamethasone (100 ng)encapsulated in poly (lactide-co-glycolide) microspheres, ova: ovalbumin(25 ug), GMCSF: granulocyte macrophage colony stimulating factor (6 ug),BSA: bovine serum albumin (25 ug). Hyperglycemia was defined as 3consecutive days with a blood glucose reading above 300 mg/dL. Six ormore mice were included in each group. Although dexamethasone blocks theaction of insulin, a controlled spatio-temporal presentation ofantigen+tolerogen led to an improvement in diabetes (greater percentageof euglycemic and slower onset of disease) in the PLGA-dex+Ova+GM-CSFgroup compared to the other groups, demonstrating that the combinationof tolerogen, antigen, and recruiting agent in the context of a scaffoldled to a reduction in a diabetes-associated autoimmune responsespecifically against pancreatic cells in vivo.

Vaccines for Attenuation of Allergic Conditions

Immunoglobulin E (IgE) is a type of antibody that is normally present insmall amounts in the body but plays a major role in allergic diseases.The surfaces of mast cells contain receptors for binding IgE. When IgEbinds to mast cells, a cascade of allergic reaction can begin. IgEantibodies bind to allergens (antigens) and trigger degranulation andthe release of substances, e.g., histamine, from mast cells leading toinflammation. Allergens induce T cells to activate B cells (Th2response), which develop into plasma cells that produce and release moreantibodies, thereby perpetuating an allergic reaction.

Scaffold-based vaccines were made to attenuate allergy, asthma, andother conditions characterized by aberrant immune activation byredirecting the immune system from a Th2 to a Th1 biased response. Thescaffold-based vaccines reduced the production of IgE that leads toallergic symptoms caused by histamine (and other pro-inflammatorymolecules) release due to mast cell degranulation.

Antibody production in response to the vaccinations was first evaluated.Balb/c mice were left untreated (No primary vaccination control). Othermice were administered 10 μg of ovalbumin incorporated into a scaffold(Ova scaffolds), 10 μg of ovalbumin with 3 μg GM-CSF incorporated into ascaffold (Ova+GM scaffolds), 10 μg of ovalbumin with 3 μg GM-CSF and 100μg CpG incorporated into a scaffold (Ova+GM+CpG scaffolds), or 10 μg ofovalbumin with 3 μg GM-CSF and 100 μg CpG injected intraperitoneally(Bolus IP (Ova, GM, CpG). Poly lactide-co-glycolide (PLG) scaffolds weremade by a gas foaming, particle leaching technique. 13 days later, theserum was collected from the animals and assayed by ELISA forova-specific IgE antibody titres. The scaffold vaccines wereadministered subcutaneously into the flank. Bolus IP injection led to anIgE antibody response. However, scaffold mediated delivery of factorsusing scaffolds (i.e., using controlled release in a spatio-temporalmanner) did not lead to an antibody response (FIG. 20). Therefore, thescaffold delivery strategy does not promote production of an allergicresponse mediated by IgE/mast cell degranulation.

On day 14, all of the mice were vaccinated with ovalbumin adsorbed toalum (adjuvant). 13 days later, serum ovalbumin-specific IgE wasquantitated (day 27). N=5-10 animals. The mice were given Ovaantigen+alum (adjuvant) to provoke a Th2-mediated allergic response. Thedata indicate that vaccination with scaffolds containingantigen+recruiting agent (GM-CSF)+Th1 promoting/stimulatory factor (CpG)reduces the Th2-mediated allergic response and preferentially increasesthe Th1-mediated response leading to reduction in allergy mediators.

The immune response elicited by the vaccines was further characterized.Balb/c mice were left untreated (No primary vaccination). Other micewere administered 10 μg of ovalbumin incorporated into a scaffold (Ovascaffolds), 10 μg of ovalbumin with 3 μg GM-CSF incorporated into ascaffold (Ova+GM scaffolds), or 10 μg of ovalbumin with 3 μg GM-CSF and100 μg CpG incorporated into a scaffold (Ova+GM+CpG scaffolds). 14 dayslater all of the mice were vaccinated with ovalbumin adsorbed to alumand 14 days later (day 28) the splenocytes from the animals werecultured with ovalbumin Media was collected from the cell culturesupernatants and IFN-gamma production or IL-4 production was assayedusing an ELISA. N=5-10 animals. The results indicated that vaccinationwith all 3 factors in a scaffold (Ova+GM+CpG scaffolds) led to anincreased level of IFN-gamma, thereby demonstrating a shift toward a Th1immune response (and away from a Th2 allergy response).

Bolus administration of CpG has sometimes been associated withsplenomegaly. Experiments were therefore carried out to evaluate spleenenlargement following vaccine administration. The results indicated thatbolus administration led to splenomegaly; however, delivery of factors(e.g., antigen/recruiting agent/Th1 stimulatory agent; Ova/GM-CSF/CpG)in a scaffold did not lead to splenomegaly. Thus, an advantage of thecontrolled spatio-temporal release of the factors from the scaffold isavoidance of the adverse side effect of spleen enlargement. Thescaffolds and methods of using them have many other advantages comparedto other strategies that have been developed to take advantage of thedendritic cell's central role in the immune system including antibodytargetting of DC and ex vivo DC adoptive transfers. The former techniquelacks specificity and unlike the scaffold poorly controls themicroenvironment where antigen is detected. Adoptive transfer is costly,ephemeral, and many of the cells die or function poorly followingadministration. The scaffold system described here is less costly,directs cells through the lifetime of the implant (continuous vs. batchprocessing), and does not require ex vivo cell processing which leads topoor cell viability and hypofunctioning.

Vaccination was evaluated in an allergy animal model of anaphylacticshock caused by and antigen trigger. Histamine release leads to a changein temperature (decrease in temperature of the subject), which was usedas a measure of the severity of allergic response. Balb/c mice wereadministered 10 μg of ovalbumin in alum (alum); 10 μg of ovalbumin with3 μg GM-CSF, and 100 μg CpG subcutaneously (bolus); 10 μg of ovalbuminwith 3 μg GM-CSF, and 100 μg CpG in a scaffold subcutaneously(scaffold); or no primary treatment (no primary) on day 0. On week 2, 5,and 8 the animals were vaccinated with ovalbumin adsorbed to alum and onweek 11 the animals were administered 1 mg of ovalbuminintraperitoneally. n=7 or 8, error bars SEM. The results shown in FIG.22 indicate that vaccination using a scaffold loaded withantigen+recruitment composition+adjuvant leads to a reduction insymptoms of allergy.

Gel Scaffold Material Based Vaccines for Treatment of Periodontitis andOther Inflammatory Dental or Periodontal Conditions

Chronic inflammation is a major component of many of dentistry's mostpressing diseases, including periodontitis, which is characterized bychronic inflammation that can lead to progressive loss of alveolar boneand tooth loss. Several tissue engineering and regeneration strategieshave been identified that may be able to reverse the destructive effectsof periodontitis, including the delivery of various morphogens and cellpopulations, but their utility is likely compromised by the hostilemicroenvironment characteristic of the chronic inflammatory state. Theinflammation in periodontitis relates to both the bacterial infectionand to the overaggressive immune response to the microorganisms, andthis has led to efforts seeking to modulate inflammation viainterference with the immune response. Therefore, there is an urgentneed to devise novel therapeutic approaches for periodontitis treatment.

Chronic inflammation is characterized by continuous tissue destruction,and is component of many oral and craniofacial diseases, includingperiodontitis, pulpitis, Sjogren's, and certain temperomandibular jointdisorders. Periodontal disease (PD), in particular, is characterized byinflammation, soft tissue destruction and bone resorption around theteeth, resulting in tooth loss. About 30% of the adult U.S. populationhas moderate periodontitis, with 5% of the adult population experiencingsevere periodontitis. Also, because PD tends to exacerbate thepathogenicity of various systemic diseases, such as cardiovasculardisease and low birth weight, PD can contribute to morbidity andmortality, especially in individuals exhibiting a compromised hostdefense. Guided tissue regeneration (GTR) membranes are commonly used toenhance periodontal regeneration, and these membranes provide a physicalbarrier to prevent epithelial cells from the overlying gingiva frominvading the defect site and interfering with alveolar bone regenerationand reattachment to the tooth. GTR membranes can enhance regeneration,although typically not in a highly predictable manner, likely due totheir passive approach to regeneration. Therefore, there is an urgentneed to devise novel therapeutic approaches for PD treatment.

One of the major complications of periodontal diseases is theirreversible bone resorption that results in the loss of affected teeth.PD is treated currently by mechanical removal of the bacteria colonizingthe teeth, and/or systemic or local antibiotic treatment. Although theseapproaches reduce the bacterial load can, when combined with appropriateoral hygiene, retard disease progression, they do not directly addressthe chronic inflammation driving tissue destruction nor promoteregeneration of the lost tissue structures. Pathogenic bone loss in PDis induced by lymphocytes that produce osteoclast differentiation factorRANKL. One approach to preventing the progression of PD leading to boneloss is to modulate T- and B-cell responses to the bacterial infectionin periodontal tissue. Using both rat and mouse models of PD, such anapproach was indeed efficient in inhibiting immune-RANKL-mediated boneresorption. The methods and compositions described herein the chronicinflammatory response must be resolved to block further tissuedestruction, and regeneration of the lost tissue must be promotedactively through inclusion of appropriate biologically active agents.

The reduce periodontal inflammation and regenerate bone previously lostto PD. For example, the pathogenic process of bone resorption andinflammation elicited by lymphocytes (FIG. 1) is suppressed by FOXP3(+)T regulatory (Treg) cells via locally activated tolerogenic dendriticcells (tDCs). After the remission of inflammatory immune response by DCthat promote the formation of regulatory T-cells (Tregs), the lost bonein the lesion is remodeled by localized delivery of a plasmid vectorwhich encodes bone morphogenic protein (BMP). The material isadministered using a minimally invasive delivery (i.e., gingivalinjection) and provides a temporally controlled release of functionallydifferent bioactive compounds. The device promotes (a) initial DCprogramming to quench inflammation via recruitment and expansion ofTregs, and (b) subsequent release of a BMP-2 encoding plasmid vector toinduce bone regeneration.

T-cells and B-cells play major role in bone resorption in PD in humanand animal models. An active periodontal lesion is characterized by theprominent infiltration of B-cells and T cells. Specifically, plasmacells constitute 50%-60% of total cellular infiltrates, which makes PDdistinct from other chronic infectious diseases. The osteoclastdifferentiation factor, Receptor Activator of NF-kB ligand (RANKL), isdistinctively expressed by activated T-cells and B-cells in gingivaltissues with PD, but not by these cells in healthy gingival tissues. TheRANKL that was expressed on the T- and B-cells in patients' gingivaltissues was sufficiently potent to induce in vitro osteoclastogenesis ina RANKL-dependent manner. The finding that RANKL is implicated as atrigger of osteoclast differentiation and activation in almost allinflammatory bone resorptive diseases emphasizes the importance ofaddressing this target.

Mouse models are recognized as the art for the study the roles of DCsand Tregs in bone regeneration processes in PD, in which inflammatoryperiodontal bone resorption is induced by the immune responses to livebacterial infection (FIG. 1). Adoptive transfer of antigen-specificT-cells or B-cells that express RANKL can induce bone loss in ratperiodontal tissue that received local injection of the T-cell antigenA. actinomycetemcomitans (Aa) Omp29 or whole Aa bacteria as the B-cellantigen. The involvement of T-cells in the bone resorption processes wasdemonstrated by two inhibitors: (1) CTLA4-Ig (binding inhibitor for Tcell CD28 binding to B7 co-stimulatory molecule expressed by APC); and(2) Kaliotoxin (blocker for T cell-specific potassium channel Kv1.3).Specifically, Kaliotoxin inhibits RANKL production by activated rat Tcells. Adoptive transfer of an Aa-specific human T-cell line isolatedfrom patients with aggressive (juvenile) periodontal disease couldinduce significant periodontal bone loss in NOD/SCID mice that wereorally inoculated with Aa every three days.

Immune responses induced to Aa-immunized mice and rats do displayPeriodontal Pathogenic Adaptive Immune Response (PPAIR). Previousstudies of rat models replicate most of the patho-physiologicalconditions of localized aggressive periodontitis (LAP) patients infectedwith Aa as well as some features of adult periodontitis. This model,relies on artificial bacterial antigen injection into gingival tissuerather than live bacterial infection. Furthermore, the lack of a varietyof gene knockout rat strains hinders elucidation of the host geneticlinkage to bacterial infection-mediated PD. A mouse model of PDreplicates many of the critical features of human PD, and the pathogenicoutcomes of adaptive immune reaction in mice, including those associatedwith RANKL induction, and is useful in terms of bone resorption inducedin the periodontal tissue.

Tregs suppress overreaction of adaptive T effector cells and quenchinflammation. Tregs were discovered originally as a subset of T-cellsthat showed suppression function in several experimental autoimmunediseases in animals. Tregs produce antigen-non-specific suppressivefactors, such as IL-10 and TGF-β. In addition, they constitutivelyexpress cytotoxic T-lymphocyte antigen 4 (CTLA-4), which down-regulatesDC activation and is a potent negative regulator of T-cell immuneresponses.

Anti-inflammatory effects mediated by Tregs also result from theup-regulation of extracellular adenosine, as Tregs convert extracellularATP to this anti-inflammatory mediator via the action of CD39 and CD73.ATP released from injured cells or activated neutrophils is implicatedas a danger signal initiator or natural adjuvant, because extracellularATP promotes inflammation. Among all lymphocyte linage cells, only Tregare reported to express both CD39 and CD73, and can also suppressadenosine scavengers. Adenosine has various immunoregulatory activitiesmediated through four receptors. T-lymphocytes mainly express the highaffinity A2AR and the low affinity A2BR. Macrophages and neutrophils canexpress all four adenosine receptors depending on their activationstate, and B-cells express A2AR. Engagement of A2AR inhibits IL-12production, but increases IL-10 production by human monocytes anddendritic cells, and selectively decreases some cytotoxic functionsmediated by neutrophils. The primary biological role of Treg appears tobe suppression of adaptive immune responses that produce inflammatoryfactors. Therefore, the ability to manipulate the formation and functionof Tregs provides novel therapeutic approaches to a number ofinflammatory immune-associated diseases, including PD (FIG. 2). Comparedto generic anti-inflammatory drugs, which require frequent dosing, it isanticipated that once Tregs are generated in sufficient numbers, theycould suppress inflammation induced by PPAIR not only in the acutephase, but also over extended time periods due to the immune memoryfunction.

Tregs are identified via their expression levels of the transcriptionfactor FOXP3. Patients with a mutated FOXP3 gene exhibit autoimmunepolyendocrinopathy (especially in type 1 diabetes mellitus andhypothyroidism) and enteropathy (characterized as ‘immunodysregulation,polyendocrinopathy, enteropathy X-linked (IPEX) syndrome’). Thesimilarity of the phenotypes between IPEX humans and Scurfy mice, whichalso show the FOXP3 gene mutation, suggests that FOXP3 mutation is acommon cause for human IPEX and mouse Scurfy. FOXP3 gene variants(polymorphism) may also be linked to susceptibility to autoimmunediseases and other chronic infections Importantly, FOXP3(+) cells arepresent in human gingival tissues, and, significantly, the expressionlevel of FOXP3 appears to diminish in diseased gingival tissue comparedto healthy gingival tissues. Even more importantly, FOXP3(+) T-cells donot express RANKL in the gingival tissues of patients who present withPD, indicating that FOXP3(+) T-cells are possibly engaged in thesuppression of PPAIR. Furthermore, the Treg-associated anti-inflammatorycytokine, IL-10, is suppressed with the expression of sRANKL in humanperipheral blood T cells stimulated in vitro by either bacterial antigenor TCR/CD28 ligation. Thus, FOXP3+ T-cells are implicated in themaintenance of periodontal health: (a) the diverse and exclusiveexpression patterns between RANKL and FOXP3 in the T-cells of humangingival tissue and (b) suppression of RANKL and other inflammatorycytokines produced by activated T-cells.

Treg cells limit the magnitude of adaptive immune response to chronicinfection, preventing collateral tissue damage caused by vigorousantimicrobial immune responses. Because periodontal disease is apolymicrobial infection, it becomes relevant to elucidate how gingivaltissue Tregs recognize such a huge and diverse variety of bacteria and,at the same time, regulate the adaptive effector T cells that also reactto a vast number of bacteria. Several lines of evidence indicate thatCD25(+)FOXP3(+)CD4(+) Treg cells are inducible from the CD25(−)CD4(+)adaptive T-cell population, especially in response to infection. Theseare often termed induced Treg cells (iTreg), and their induction, whichis remarkably similar to the naturally-occurring Treg (nTreg)populations, is generated by peripheral activation, particularly in thepresence of IL-10 or TGF-β. The diversity of T-cell receptors (TCRs)within the whole FOXP3(+) Treg population exceeds that of FOXP3(−)CD4 Tcells. The presence of antigen-specific Treg has also been found in avariety of infectious diseases, including Leishmania, Schistosoma, andHIV. All these results are consistent with the mechanism that Tregrecognize foreign antigens. Because periodontal disease is apolymicrobial infection, it becomes relevant to utilize Treg insuppressing the inflammation associated with the activated adaptiveeffector T-cells that also react to a vast number of bacteria.

The immune response (e.g., Treg induction) is orchestrated by a networkof antigen-presenting-cells, and likely the most important of these celltypes are DCs. Tissue-resident DCs routinely survey and capture antigen,and present antigen fragments to T-cells. The antigen presentation byDCs plays a key role in directing the immune response against theantigen to either immune activation or tolerance. In the healthygingival tissue, immune tolerance against the oral commensal bacteria isinduced, whereas immune activation is elicited to the periodontalpathogens in the context of PD, as demonstrated by elevated IgG antibodyresponse to the periodontal pathogens, as described above. These twoopposed outcomes, tolerance vs. activation, are controlled by the DCspresent in the gingival tissue. Tolerance-inducing DCs (tDCs) are alsocalled regulatory DCs. One method used by tDC to prevent immuneactivation is to generate iTreg cells during antigen presentation. Thestate of maturation and activation of DCs is critical to Tregdevelopment: DCs activated and maturing in response to inflammatorystimuli trigger immune responses, but immature or “semimature” DCs, incontrast, induce tolerance mediated by the generation of Tregs. Themajor phenotypic feature of tDC is their production of IL-10 and low orno production of IL-12 and other cytokines that prime effector T-cells.A number of signals and cytokines direct DC trafficking and activation.Multiple inflammatory cytokines mediate DC activation, including TNF,IL-1, IL-6, and PGE2, and are frequently used to mature DC ex vivo.

Granulocyte macrophage colony stimulating factor (GM-CSF) is aparticularly potent stimulator of DC recruitment and proliferationduring the generation of immune responses, and is useful to manipulateDC trafficking in vivo. A variety of exogenous factors including TGFβ,thymic stromal lymphopoietin (TSLP), vasoactive intestinal peptide(VIP), and retinoic acid (RA), used alone or in combination, orientateDC maturation induce tolerance, and Treg development.

Morphogens

A number of morphogens (e.g., bone morphogenetic proteins (BMPs),platelet derived growth factor (PDGF)) that actively promote boneformation by tissue resident cells are useful for prompting alveolarbone regeneration. The BMPs, members of the TGF-β superfamily, play akey role in that process. The BMPs are dimeric molecules that have avariety of physiologic roles. BMP-2 through BMP-8 are osteogenicproteins that play an important role in embryonic development and tissuerepair. BMP-2 and BMP-7, the first BMPs to be available in a highlypurified recombinant form, play a role in bone regeneration. BMP-2 actsprimarily as a differentiation factor for bone and cartilage precursorcells towards a bone cell phenotype. BMP-2 has demonstrated the abilityto induce bone formation and heal bony defects, in addition to improvingthe maturation and consolidation of regenerated bone. PDGF is a proteinwith multiple functions, including regulation of cell proliferation,matrix deposition, and chemotaxis, and has also been investigated forits potential to promote periodontal regeneration. PDGF deliveryinfluences repair of periodontal ligament and bone, and ligamentattachment to tooth surfaces. Recombinant proteins are used as theactive agent in bone regeneration therapies. Alternatively local genetherapy strategies are used to deliver morphogen.

Sustained local production and secretion of growth factors via genetherapy overcomes certain limitations of protein delivery related toshort half-life and susceptibility to the inflammatory environment, andalso allows regulation of the timing of factor presence at a tissuedefect site. Small-scale clinical trials and animal studies havedocumented success utilizing adenovirus gene delivery approaches, ortransplantation of cell populations genetically modified in vitro priorto transplantation, to promote local expression of growth factors todrive bone regeneration. Delivery of plasmid DNA containing genesencoding for growth factors is preferred. Plasmid delivery requireslarge doses, and this results in expression of the transgene for about 7days or fewer. Plasmid DNA delivery from polymer depots, increasestransfection efficiency and duration of morphogen delivery.

Delivery Systems

Programming of DCs and host osteoprogenitors in situ to generate potent,and specific immune and osteogenic responses involves preciselycontrolling in time and space a variety of signals that act on thesecells. One approach to provide localized and sustained delivery ofmolecules at the desired site of action is via their encapsulation andsubsequent release from polymer systems. Using this approach, themolecule is slowly and controllably released from the polymer (e.g., viapolymer degradation), with the dose and rate of delivery dependent onthe amount of drug loaded, the process used for drug incorporation, andthe polymer used to fabricate the vehicle. In addition, polymer systemspermit externally regulated release of encapsulated bioactive moleculese.g., using ultrasound as the external trigger. A variety of differentpolymers, and varying physical forms of the polymers have been developedto allow for localized and sustained delivery of various bioactivemacromolecules. Biodegradable polymers of lactide and glycolide (PLG),which are also used to fabricate GTR membranes, are used clinically forextended delivery of hormones (Lupron Depot® microspheres [TakedaChemical], and Zoladex microcylindrical implants [ZenecaPharmaceuticals]. PLG microspheres that sustain the release ofMacrophage Inflammatory Protein (MIP-3β) are chemoattractive for murinedendritic cells in vitro. Polymer rods have also been used to locallycodeliver MIP-3β with tumor lysates or antigen, and induced therecruitment of dendritic cells that were able to induceantigen-specific, cytotoxic T-lymphocyte activity that yieldedanti-tumor immunity.

Intratumoral injection of GM-CSF and IL-12 loaded microspheres was shownto generate protective immunity. Alginate-derived polymer, a depotsystem suitable has been used as carrier for immune regulating cues andosteogenic stimuli. Alginate is a linear polysaccharide comprised of(1-4)-linked β-D-mannuronic acid and α-L-guluronic acid residues, and ishydrophilic. Alginate gels promote very little non-specific proteinabsorption, likely due to the carboxylic acid groups, and has anextensive history as a food additive, dental impression material, and ina variety of other medical and non-medical applications. In the pureform, it elicits very little macrophage activation or inflammatoryresponse when implanted Sodium salts of alginate are soluble in water,but will gel following binding of calcium or other divalent cations toyield gels that may readily be introduced into the body in a minimallyinvasive manner. These material systems have the ability toquantitatively control DC trafficking in vivo, and to specificallyregulate DC activation. Such material systems provide control of hostimmune and inflammatory responses, while simultaneously providingsignals that actively promote periodontal tissue regeneration.

Chronic Inflammation in Periodontal Diseases (PD)

Chronic inflammation accompanying PD promotes bone resorption viainvolvement of immune cells (FIG. 1). Materials, hydrogels inparticular, and therefore introduced into diseased tissue and firstdeliver signals to alter the balance of the immune response toameliorate inflammation, and subsequently provide on-demand, localizeddelivery of pDNA encoding BMP-2. These compositions and methods lead tosignificant bone regeneration (FIG. 2). DCs are targeted as a centralorchestrator of the immune system, are potent antigen-presenting cells.Other cell types may provide targets for immune modulation, and thestrategies described herein are applicable to those cell types as well.This invention provides for material systems that program DCs in orderto alter the balance between Tregs and effector T-cells to amelioratechronic inflammation. The ability of Tregs to produce anti-inflammatorycytokines such as IL-10, and suppress adaptive immune responses makesthem an attractive target to ameliorate chronic inflammatory processes.Material systems offer the opportunity to control more precisely thenumbers, trafficking, and states of DCs and T-cells in the body, incombination with their ability to provide osteoinductive stimuli.

In another aspect of the invention, bone regeneration is promoted via aninductive approach that involves localized delivery of plasmid DNAencoding BMP-2. Local gene therapy is used to promote osteogenesis, andpDNA approaches in particular. The therapeutic system combinesosteoinductive factor delivery with the active quenching ofinflammation, and the externally-triggered release of the osteoinductivefactor once inflammation is diminished. In particular embodiments,alginate hydrogels are used as the material platform. These gels areintroduced into the body in a minimally invasive manner and have provenuseful to deliver proteins, pDNA and other molecules, and regulate theirdistribution and duration in vivo. Alginate hydrogels are particularlyuseful for the ultrasound-mediated triggered release.

Further regarding the material system to recruit large numbers of hostDCs and to effectively induce these DCs to a tolerant state (tDCs),GM-CSF are a cue to recruit DCs and TSLP pushes recruited DCs to the tDCphenotype. The GM-CSF is released into the surrounding tissue to recruitDCs, promote their proliferation, and generally increase the numbers ofimmature DCs, while appropriate TSLP exposure converts these cells totDCs. The relation between local GM-CSF and TSLP delivery and tDCs,leads to generation of tDCs while minimizing the numbers of activatedDCs.

One embodiment characterizes the action of GM-CSF and TSLP, and theirdelivery via alginate gels. GM-CSF is a potent signal for DC recruitmentand proliferation, and the GM-CSF concentration is key to its ability toinhibit DC maturation and induce tolerance. TSLP generates tDCs due toits ability to initiate and maintain T-cell tolerance. A number of otherfactors have been identified that enhance formation of tDCs and Tregs,including vasoactive intestinal peptide, Vitamin D and retinoic acid,and these may be used alone or in combination with TSLP.

Materials containing the GM-CSF and TSLP with the appropriatespatiotemporal presentation to recruit and develop tDCs in situ weredeveloped. The effects of continuous GM-CSF and TSLP exposure (10-500ng/ml GM-CSF; 10-200 ng/ml TSLP) are described herein. FACS analysis andother analytic method used are to characterize DC population by deletingmarkers of maturation, e.g. MHCII, CD40, CD80 (B7-1), CD86 (B7-2), andCCR7, evaluating their secretion of cytokines (TNF-α, IL-6, IL-12,IFN-α, IL-10 tDC are identified by low levels of CD40, CD80, CD86,MHCII, and high level of IL-10). The effects of gradients of GM-CSF oncell recruitment is evaluated using a diffusion chamber.

Alginate gels with varying rheological/mechanical properties anddegradation rates are created through control over the polymercomposition, molecular weight distribution, and extent of oxidation. Thealginate formulation used was binary alginate composed of 75% oxidizedlow molecular MVG alginate and 25% high molecular weight MVG alginatecrosslinked with calcium. The scaffold compositions allows the localizeddelivery of GM-CSF and TSLP. The release rates of GM-CSF and TSLPdepends on the gel cross-linking and degradation rate, e.g., the gelsprovide sustained release for a time-frame ˜1-2 weeks. These moleculesare incorporated directly into the gel during cross-linking, asdocumented previously for other growth factors and pDNA. If the releaseoccurs too rapidly (e.g., gel depleted within 1-2 days), the release maybe retarded by first encapsulating the factors in PLG microspheres, thatare then incorporated into gels, alginate gels, during cross-linking. Inthis approach, release from the PLG particles regulates overall release,and this rate is tuned by altering the MW and composition of the PLG.The release rates of the GM-CSF and TSLP are analyzed in vitro usingiodinated factors, following factor encapsulation. For example, GM-CSFis released over a period of 2 days to 3 weeks. The bioactivity of thereleased factors is confirmed using standard cell-based assays known inthe art.

Gels are injected in the gingival tissue of mice at the site of alveolarbone loss (e.g., 1.5 μl).

The ability of GM-CSF and TSLP to recruit host DCs (FIG. 4) indicatesthat an appropriate GM-CSF dose ranges from 200 ng-10,000 ng. Thefollowing factors were used to evaluate.

Mouse Cytokine/Chemokine Panel-24-Plex

Cytokine Chemokine Chemokine receptor(s) TNF-a Eotaxin CCR3 G-CSF IP-10CXCR3, CXCR3B GM-CSF KC CXCR2 M-CSF MCP-1 CCR2** IFN-γ MIG CXCR3 IL-1βMIP-1a CCR1, CCR5** IL-2* MIP-1β CCR5** IL-4* MIP-2 CXCR2 IL-6 RANTESCCR1, CCR3, CCR5** IL-7* IL-9* IL-10 IL-12 (p70) IL-15* IL-17*γc-receptor-dependent cytokines **reported to be expressed on Treg

Presentation of GM-CSF yields large numbers of recruited DCs, and acorrelation between GM-CSF concentrations and DC maturation obtained(e.g., DCs maturation be inhibited at high GM-CSF concentrations). Inother words, by controlling the release kinetics and dose of GM-CSF, itcan act not only as a recruiting factor, but a tolerogenic factor. Forexample, at high concentrations of GM-CSF dendritic cells can becometolerogenic. If insufficient numbers of DCs are recruited with GM-CSF,exogenous Flt3 ligand release from gels is optionally used. TSLP iscritical to direct the activation of DCs, particularly in the presenceof inflammatory signals (e.g., LPS). The dose of TSLP relative to GM-CSFcontributes to this phenomena. For example, the range for each factor ina scaffold is 0.1 μg to 10 μg, e.g., scaffolds were made using 1 μg ofeach. TGF-beta, IL-10, rRetinoic acid, Vitamin D, and/or vasoactiveintestinal peptide can optionally be added or used in place of TSLP.Alginate or PLG are preferred polymers; however other polymers andmethods of TSLP and GM-CSF immobilization within the gels are known inthe art.

Modulating PD-related inflammation with materials presenting GM-CSF andTSLP induces the formation of Treg cells and ameliorates inflammation inmice with PD. Inflammatory bone resorption found in human patients withPD was shown to be elicited by activated adaptive immune T-cells (andB-cells) which produce bone destructive RANKL as well as collateralinflammatory damage caused by expression of proinflammatory cytokines(IL-1-β, IFN-γ) from T-cells and other accompanying inflammatory cells.Suppressing the activation of T cells resolves the chronic inflammationand bone resorption associated with periodontal disease. Locallyinducing anti-inflammatory Treg cells (iTregs) using the GM-CSF/TSLPmaterial gel system shows tDCs generated by GM-CSF and TSLP formation ofiTregs and inhibit the inflammatory bone resorption induced byactivation of adaptive immune responses. The level of inflammation ismonitored by measurement of inflammatory chemical mediators present ingingival tissue (PGE₂, nitric oxide, ATP and adenosine) and presence ofinflammatory cells.

Induction of tDCs in Periodontal Disease

The PD mouse model induces vertical periodontal bone loss followingactivation of immune responses to orally harbored bacteria, termed“Periodontal Pathogenic Adaptive Immune Response (PPAIR)”. Vertical boneloss is most closely associated with the human form of periodontaldisease, and this PD model permits evaluation of: (1) inflammatoryresponse by measurement of proinflammatory cytokines in the tissuehomogenates; (2) localization and number of FOXP3+ Treg cells usingFOXP3-EGFP-KI mice; (3) phenotypes of inflammatory cells by triple-colorconfocal microscopy and flow cytometry; (4) presence of bone destructiveosteoclasts (TRAP), bone-generating osteoblasts (Periostin/alkalinephosphatase [ALP]), and ligament fibroblasts (Periostin/ALP); and (5)the level of bone resorption. Instead of a membrane-based GTR system,the selection of a gel-based delivery system is useful as a minimallyinvasive (non-surgical) material system to remodel vertical bone loss.More specifically, one gingival injection of gel appropriately deliversGM-CSF/TSLP. The socket wall at the vertical bone resorption lesionprovides the space to retain the material, without the aid of ascaffold. After the successful demonstration of the principlesunderlying this approach, these gels are used as a supplement to currentmembrane-based GTR systems, or GTR systems that similarly provide thesecues could be developed.

It is striking that increased numbers of FOXP3+ Treg cells were observedalong with IL-10+CD11c+DC cells in the mouse periodontal bone losslesion where GM-CSF/TSLP-gel was injected (FIG. 9). These data indicatethat tDCs enhance local enrichment of (or promote generation of) FOXP3+Treg cells. The GM-CSF/TSLP-delivered gel to induce tDCs. These aspectsshows the kinetics of iTreg induction by GM-CSF/TSLP delivery inalginate gels in periodontal bone loss lesions. The impact of the localformation of iTreg cells on the bone remodeling system (i.e.,osteoclasts vs. osteoblasts and ligament fibroblasts) and continuationof bone resorption was observed.

GM-CSF enhanced DC recruitment and proliferation in a dose-dependentmanner (FIG. 3A-3B). High concentrations (>100 ng/ml) of GM-CSF,however, inhibited DC migration toward the LN-derived chemokine CCL19(FIG. 3C) Immunohistochemical staining revealed that the highconcentrations of GM-CSF also suppressed DC activation via TNF-α and LPSstimulation by down-regulating expression of MHCII and the CCL19receptor CCR7 (FIG. 3D). These results indicate that local, high GM-CSFconcentrations recruit large numbers of DCs and prevent their activationto a phenotype capable of generating a destructive immune response.

The GM-CSF/TSLP the recruitment of DCs and subsequent activation ofiTregs, and provides local, material-based delivery of pDNA encodingosteogenic molecules in vitro leading to bone regeneration.

The polymer delivery vehicle presents GM-CSF in a defined spatiotemporalmanner in vivo, following introduction into the tissue of interest.Exemplary vehicle quickly release approximately 60% of the bioactiveGM-CSF load within the first 5 days, followed by slow and sustainedrelease of bioactive GM-CSF over the next 10 days (FIG. 4A), to allowdiffusion of the factor through the surrounding tissue and effectivelyrecruit resident DCs. Polymers were loaded with 3 μg of GM-CSF andimplanted into the dorsal subcutaneous site of C57BL/6J mice.Histological analysis at day-14 revealed that the total cellularinfiltration at the site was significantly enhanced compared to control(no incorporated GM-CSF) (FIG. 4B). FACS analysis for CD11c(+)CD86(+)DCs showed that GM-CSF increased not just the total cell number, butalso the percentage of infiltrating cells that were DCs (FIGS. 4C-4D).Enhanced DC numbers at the material-implanted site were sustained overtime (FIG. 4E). As predicted by in vitro testing, the effects of GM-CSFon in vivo DC recruitment were dose-dependent (FIG. 4F).

The present invention provides for a material-based local application ofGM-CSF with appropriate DC influencing factors that leads to tolerogenicDCs (tDCs), and subsequent enrichment of iTreg cells. Candidatebiofactors include thymic stromal lymphopoietin (TSLP), vasoactiveintestinal peptide (VIP), and transforming growth factor-beta (TGF-β).Screening is based on the induced DC's anti-inflammatory properties. Thein vitro incubation of mononuclear cells isolated from the bone marrow(BM) of C57BL/6 mice with GM-CSF in the presence of TSLP, VIP, or TGF-βled to diminished expression of the proinflammatory cytokines IL-6 andIL-12, in response to bacterial stimulation, as compared to the DCinduced by GM-CSF alone (FIG. 5). In response to bacterial challenge,however, GM-CSF/TSLP-induced DC produced the highest levels of theanti-inflammatory cytokine, IL-10, as compared to the othercombinations. Interestingly, the addition of TSLP did not alter theyield of GM-CSF-mediated differentiation of DC(CD11c+/CD86+ in total BMcells; GM-CSF alone, 14.7% vs. GM-CSF+TSLP, 14.6%) from the BM cellscompared to the low yield of CD11c+/CD86+DC with TGF-b (10.5%)(FIG. 5,Table 1). Overall, these observations that the combination of GM-CSFwith TSLP efficiently induces DC with an anti-inflammatory phenotype.

To demonstrate that material-based delivery of GM-CSF/TSLP inducestolerogenic DC locally in vivo, polymer vehicles containing a mixture ofGM-CSF (1 μg) and TSLP (1 μg), as well as GM-CSF alone (1 μg), wereinjected into the periodontal bone resorption socket of FOXP3-EGFP-KImice (C57BL6 background), and were evaluated to determine their effectson the local DC cells. Seven days later, a remarkable increase in theproportion of CD11c+IL-10+ DC was observed in the periodontal socket ofmice receiving polymers containing GM-CSF/TSLP, as compared to theinjection of control empty polymer (FIG. 6). These findings indicatethat the local delivery of TSLP and GM-CSF by the polymer can positivelyskew the GM-CSF-mediated differentiation of DC with anti-inflammatoryactivity, represented by high IL-10 expression, in the previouslydeveloped periodontal bone resorption lesion.

The ability of the material systems of the present invention not only torecruit DCs, but also to regulate T-cell generation, was also examined.These studies were performed to elicit an anti-tumor immune responseagainst melanoma via inclusion in the material of “DC activators”(cytosine and guanosine-rich oligonucleotides; CpG-ODN; TLR9 ligand thatelicits danger signal in DC, and melanoma-specific antigen, along withthe GM-CSF. Nevertheless, although such approach “to activate immuneresponse” contradicts to the approach “to suppress inflammatory-immuneresponse,” the results demonstrate the ability to generate specific andquantitative immune responses with the material systems. Specifically,over 17% of the total cells at the site were CD8(+) compared to thecontrol non-treated site (<1% CD8) (FIG. 6A). This result indicates thatthe number of T-cells infiltrating tissue adjacent to the polymericdelivery vehicle was enriched with delivery of GM-CSF, antigen andCpG-ODGN. The generation of a specific memory immune response was shownby staining isolated splenocytes with MHC class I/tyrosinase-relatedprotein (TRP2). This analysis revealed a significant expansion ofTRP2-specific CD8 T-cells in mice vaccinated with GM-CSF, antigen andCpG-ODN (0.55% splenocytes, 1.57×10⁵+5.5×10⁴ cells) in comparison tomatrices presenting lower CpG doses, either 0 μg or 50 μg (0.17% and0.25% of splenocytes) (FIG. 6B). As indicated above and in the nextsection (FIG. 10), the findings that the materials delivering GM-CSF andCpG oligonucletides activate anti-tumor CD8 T-cells by activation of DCexpressing IL-12, and in contrast when delivering GM-CSF and TSLPactivate Treg cells by activation and differentiation of tolerogenic DCthat produce IL-10, confirm the power of this approach to regulateimmune responses.

The mouse model of PD was also used to study the efficacy of minimallyinvasive material systems that can suppress PPAIR, as well as induceregeneration in the bone loss lesion of PD, which meets theimmuno-pathological fundamentals found in humans. This model developsRANKL-dependent periodontal bone loss upon induction of adaptive immuneresponses to the mouse orally colonized bacteria. By using the 16S rRNAsequence method, it was discovered herein that in-house bred BALB/c miceharbor the oral commensal bacterium Pasteurella pneumotropica (Pp). Ppis facultative anaerobic Gram(−) bacterium, and, similar to Aa, Pp isresistant to Bacitracin and Vancomycin, but susceptible to Gentamycin.Aa and Pp, as well as Haemophilus, belong to the same phylogenic familyof Pasteurellaceae. Pp outer membrane protein OmpA is a homologue of AaOmp29. Natural oral colonization of BALB/c mice with Pp per se is latentand has not shown any pathogenic features because immunologicaltolerance is induced to this oral commensal Pp. Supporting this,Pasteurella was also reported to be commensal in the gingival crevice offerrets. Thirty days after either (1) adoptive transfer of theAa-reactive Th1 line; or (2) peripheral immunization (dorsal s.c.injection) with fixed whole Aa to the Pp-harboring mice, periodontalbone loss (horizontal) was demonstrated, along with elevated IgGantibody response to Aa Omp29, and increased production of TNF-α andRANKL in the gingival tissue. The T-cells infiltrating in the gingivaltissue expressed RANKL in the group of PD-induced mice, but not in thecontrol group. Furthermore, systemic administration of OPG-Fc inhibitedthe periodontal bone loss induced in this mouse PD model, indicatingthat the induced periodontal bone loss is RANKL-dependent. The Aaimmunization to the “Pp-free” BALB/c mice did not show periodontal boneloss, indicating that orally colonized commensal Pp bacteria thatdeliver the T-cell antigen to mouse gingival tissues is required forbone loss induction. Serum IgG of Aa-immunized Pp+ mice reacted to bothAa and Pp, but not other oral bacteria or E. coli examined. This verydistinct cross-reactivity between Aa Omp29 and Pp OmpA allows theinduction of PPAIR that results in periodontal bone loss by immunizationof Pp+ mice with Aa antigen. Indeed, Omp29 is one of the most prominentantigens recognized by serum IgG antibody in LAP patients infected withAa.

Although mouse models of P. gingivalis oral infection have been mostfrequently investigated, these P. gingivalis infection models appear todisplay mechanisms different from PPAIR. This occurs because inductionof adaptive immune responses displayed by elevated IgG antibody to P.gingivalis antigen ameliorates, instead of augments, the P.gingivalis-infection-mediated periodontal bone loss, which is notnecessarily representative of human periodontal bone resorption. Anothershortcoming of the P. gingivalis-induced mouse PD model derives from theinduction of only “horizontal periodontal bone loss,” while human PD ischaracterized by both “horizontal” and “vertical” periodontal bone loss.Although a number of etiological causes are proposed, horizontal boneloss is said to occur when chronic periodontal disease progressesmoderately, while vertical bone loss is indicated when severe recurrentperiodontitis or severe acute periodontitis progresses. The differenceis important in the context of the proposed study because, while“horizontal” periodontal bone loss can be maintained by non-surgicalperiodontal treatment, “vertical bone loss” is, in fact, the clinicalcase where GTR surgery is required (FIG. 8).

Vertical periodontal bone loss with inflammatory connective tissue inmouse PD model, using the C57BL/6 strain mice, which followed the sameprotocol as published for BALB/c strain, demonstrated massiveirreversible “vertical” periodontal bone loss (FIG. 7). This mirrors theperiodontal bone loss found in most human patients with severe PDbecause, once having developed, vertical bone loss remains, even afterthe resolution of severe inflammation. For example, bone decay at thetooth extraction socket of mice is completely filled with new bonewithin 15 days. In contrast, vertical bone loss induced by PPAIRremains, indicating a significant difference in bone regenerationprocesses between bone loss caused by tooth extraction and by PD. It isnoteworthy that few of the previously published animal models of PDdevelop vertical periodontal bone loss, and most of the periodontal boneloss induced in these animal models seems to develop horizontally and tobe reversible after the resolution of inflammation. Therefore, thisnewly established mouse model, provides the ideal platform with which toevaluate minimally invasive material systems that down-regulateinflammation as well as induce regeneration of lost bone. As illustratedin FIG. 7 (7 g: control; 7 h: PD lesion), the PD mice develop verticalbone loss filled with inflammatory connective tissue accompanied byTRAP+ osteoclast cells. Thus, minimally invasive material systems, suchas the GM-CSF/TSLP delivery polymer described herein, can beadministered to the inflammatory bone loss lesion such that bothinflammatory response and bone regeneration in the bone loss lesion canbe evaluated.

Adoptive transfer of FOXP3+ CD4 T cells inhibits in vivo mouse boneresorption induced by PPAIR. In order to investigate if an increase ofFOXP3+ Treg cells can suppress PPAIR-caused periodontal bone resorption,CD25+FOXP3+CD4+ iTreg cells were isolated from spleen T cells stimulatedwith TGF-b, IL-2 and Aa-antigen (FOXP3+CD25+ cells were 79.8% of thetotal CD4 T-cells) and were adoptively transferred to Pp+ BALB/c micethat were immunized with fixed Aa (dorsal s.c.) on Day-0, -2 and -4. Inan in vitro assay, CD25+FOXP3+CD4+ iTreg cells suppressed theproliferation and production of RANKL by antigen/APC-stimulatedAa-specific Th1 effector cells (FIG. 8B). For control, non-immunizedmice and Aa-immunized mice, without adoptive transfer, were prepared.Thirty days after Aa immunization, PPAIR was observed in theAa-immunized mice, as determined by the elevated IgG1 responses toOmp29, elevation of IFN-γ and sRANKL in the local gingival tissue (FIGS.8D and 8E), and periodontal bone resorption (FIG. 8C). The transfer ofCD25+FOXP3+CD4+ iTreg cells to mice that received Aa systemicimmunization significantly inhibited the following PPAIR features ascompared to positive control animal groups: (1) increased IgG1 responsesto Omp29; (2) IFN-g and sRANKL concentration in the gingival tissue(FIGS. 8D and 8E); and (3) local periodontal bone resorption (FIG. 8C).The amount of anti-inflammatory cytokine IL-10 in the gingival tissuewas significantly increased by the transfer of iTreg cells (FIG. 8F).These results strongly suggest that local expansion of CD25+FOXP3+CD4+iTreg cells can, in fact, inhibit periodontal inflammatory boneresorption induced by PPAIR by the mechanism of suppression of sRANKLand IFN-γ while activating IL-10 production in the local gingivaltissues. This finding may be important in the context of the presentinvention because the efficacy of a material system in suppressingperiodontal inflammation may be generated not by adoptive transfer, butby increasing host iTreg cells via activation of tolerogenic DC.

Local injection of polymer delivering GM-CSF/TSLP increases FOXP3+T-cells in mouse gingival tissue and local lymph nodes (LN). Theinjection of polymeric delivery vehicles into the periodontal boneresorption socket of PD-induced FOXP3-EGFP-KI mice (C57BL6 background)was evaluated for the effects of the polymer on the resultantproportionality of Treg cells in the periodontal bone resorption lesionas well as local (cervical) lymph nodes. Seven days after the injectionof polymer containing a mixture of GM-CSF (1 μg) and TSLP (1 μg) intothe periodontal bone resorption socket (bone loss lesion developed 30days after PPAIR induction by fixed Aa injection), an increase wasobserved in the proportion of FOXP3+EGFP+ Treg cells in cervical lymphnodes of mice that received GM-CSF/TSLP delivery polymer, whereasinjection of polymer with GM-CSF (1 μg) alone did not show such increaseof FOXP3+EGFP+ Treg cells in the local lymph nodes compared to thecontrol empty polymer injection (FIG. 9). Interestingly, in theconnective tissue of PD lesion, remarkable infiltration of FOXP3+ cellswas observed in the mice receiving GM-CSF/TSLP-polymer, as well asGM-CSF-polymer, while few FOXP3+ cells were detected in the bone losslesion of mice that did not receive any injection. Of interest, theFOXP3+ cells were found in foci that are composed of a number ofinflammatory cell infiltrates, suggesting that the injected polymer mayprovide a scaffold for Treg cells to react with tolerogenic DC. Tosupport this premise, the co-localization of FOXP3+ cells andtolerogenic DC was observed in the legion that receivedGM-CSF/TSLP-polymer (FIG. 9C). Therefore, the GM-CSF/TSLP polymermaterial delivery system demonstrably expanded the anti-inflammatoryFOXP3+ Treg cells in periodontal bone resorption lesion as well as locallymph nodes.

Materials for localized pDNA delivery and tissue regeneration, andpolymer systems for sustained pDNA release were developed to allow forthe localized delivery and sustained expression of pDNA with kineticsdependent on the rate of polymer degradation. Macroporous scaffolds ofPLG may be used for the encapsulation of pDNA, with its subsequentrelease regulated by the degradation rate of the particular PLG used forencapsulation; allowing for sustained release of plasmid DNA for timesranging from 10-30 days. To enhance the uptake of pDNA, and to localizethe plasmid to the region encompassed by the polymer, pDNA was condensedwith PEI prior to incorporation into the polymeric vehicles Implantationof scaffolds containing either an uncondensed or PEI-condensed markergene (luciferase) resulted in the short-term expression of theuncondensed DNA, but a very high and extended duration of expression forthe PEI-condensed DNA. Further, implantation of polymers delivery PEIcondensed pDNA encoding for BMP-2 or BMP-4 led to long-term BMP-4expression by host cells (FIG. 10A), and significantly more boneregeneration than the polymer alone, delivery of non-condensed pDNA, orno treatment (FIG. 10B-10D).

This approach can be extended to injectible alginate gels. Thedegradation rate of alginate gels is altered by controlling themolecular weight distribution of the polymer chains comprising the gels.The rate of gel degradation (FIG. 11A) strongly correlated with thetiming of release of PEI condensed pDNA encapsulated in the gels (FIG.11B). The timing of pDNA expression in vitro and in vivo was regulatedby the gel degradation rate, and this approach to pDNA delivery led tophysiologically relevant expression in vivo of an encoded morphogen, andsignificant effects on local tissue regeneration.

The present invention provides for the delivery of pDNA encoding anosteogenic factor subsequent to amelioration of chronic inflammation,using regulated pDNA release from the delivery vehicle. Ultrasoundirradiation may be used to trigger the release of pDNA from alginatehydrogels, as ultrasound may provide an external trigger to controlrelease of drugs from materials placed in periodontal tissue. Ultrasoundhas been pursued widely in past studies of drug delivery from theperspective of permeabilizing skin to enhance drug transport, but inpresent invention exploits the transient disruption of the gel structureduring ultrasound application to enhance release of pDNA encapsulated inthe gels. Use of a high molecular weight, non-oxidized alginate to formthe gel (unary gel in FIG. 12A) led to minimal background release ofpDNA, due to the slow degradation of this gel (FIG. 12). Application ofappropriate ultrasound irradiation led to a 1000-fold increase in thepDNA release rate; the rate rapidly returned to baseline levelsfollowing cessation of irradition (FIG. 12). The increase in pDNArelease with ultrasound application correlated with large-scaleperturbations of gel structure, as noted in past studies for biologicalsamples. The subsequent rapid return of pDNA release rate to base-linelevels correlated with a reversal of the gel structure to the originalstate. The ability of the alginate gels to “heal” following ultrasoundlikely is due to their reversible cross-linking with calcium ions intheir environment. The present invention thus provides for precisecontrol the timing of release of pDNA encoding osteogenic stimuli fromthe biomaterials matrix, at a time-point sufficient to first allow forconversion of the immune response to a non-inflammatory state.

Analysis of Kinetics of Gingival Treg Cell Induction in the Mouse PDModel

Experiments were carried out to determine how long it takes for theinduction of Treg cells and alterations in the local inflammatoryenvironment with GM-CSF/TSLP delivery by alginate gel. Knowing theoptimal time when inflammation is sufficiently and efficiently quenchedby GM-CSF/TSLP-gel injection indicates the optimal timing for therelease of pDNA-encoding BMP2 from the material system.

FOXP3-EGFP-KI mice (8 wk old, 12 males/group) that harbor Pp in the oralcavity receive immunization of fixed Aa (10⁹ bacteria/site/day dorsals.c. injection on Day 0, 2 and 4). At Day-30, the development ofperiodontal bone loss is confirmed by probing of gingival pockets ofmaxillary molars. Serum IgG responses to Pp and Aa, along with thecross-reactive immunogenic antigens, including Pp OmpA (a homologue ofAa Omp29), are measured by ELISA because elevated IgG response to Ppantigens at Day-30 confirms that PPAIR successfully induces thedevelopment of vertical bone loss. Assuming that the levels of bone lossbetween left and right sides at Day 30 are symmetrical in each animal,the effects of GM-CSF/TSLP and the role of induced Treg cells areevaluated by palatal maxillary injection of gel with and withoutCD25+FOXP3+ Treg depletion by anti-CD25 MAb:

Group A: an injection of (1) mock empty gel to left, and 2) GM-CSF/TSLPto right, palatal maxillary gingivae;

Group B: same gingival injections as Group A, but the mice receiveanti-CD25 MAb (500 μg/mouse, i.v. rat MAb hybridoma clone PC61 fromATCC) 3 days prior to gel injection;

Group C: same gingival injections as Group A, but the mice receivecontrol purified rat IgG (500 μg/mouse, i.v.) 3 days prior to the gelinjection;

Group D: an injection of mock empty gel to left, but no injection to theright, palatal maxillary gingivae.

The alginate gels were injected into the bone loss legion (1.5 μl/site).Animals are sacrificed on Day-33, -37, -44, and -58 (=3, 7, 14 and 28days after injection of gels, respectively). Control, non-treatedC57BL/6 mice sacrificed on Day-30 provide base-line information aboutinflammatory response and level of bone loss before the treatment withGM-CSF/TSLP-gel. The depletion of CD25+FOXP3+ Treg cells in Group B isconfirmed by detection of CD25+FOXP3+ cells in the peripheral bloodisolated from Group B and Group C using flow cytometry at Day-30. Thedose and timing of TSLP/GM-CSF presentation from gels is determined, and2-3 different doses are tested. Analysis included of: (1) Fluorescentimmunohistochemistry for the detection of FOXP3+EGFP+ Treg cells andother inflammatory cell types (e.g., macrophages, neutrophils), gingivaltissue cytokine measurement, detection of inflammatory chemicalmediators in gingival tissue, and measurement of FOXP3+EGFP+ Treg cellsand other lymphocyte phenotypes in cervical lymph nodes by flowcytometry; (2) analyses of TRAP+ osteoclasts, Periostin+/ALP+osteoblasts and Periostin+/ALP+ ligament fibroblasts in decalcifiedperiodontal tissues; and (3) extent of bone resorption using micro-CT,and quantitative histomorphometry.

Evaluation of Effects of GM-CSF/TSLP-Gels on the Immune Memory of iTregResponse

The efficacy of gel delivery of GM-CSF/TSLP in eliciting immune memory,as challenged by recurrent activations of PPAIR, was explored. Theaspect of immune memory is significant because once immune memory ofiTreg response can be induced, it should be capable of preventingrecurrent episodes of pathogenic periodontal bone loss at the same site,and the development of future periodontal bone loss at different sites.

PD was induced as described above. At Day-30, Groups A and B receiveidentical gingival injections: (1) an injection of mock empty gel toleft, and (2) an injection of GM-CSF/TSLP to right, palatal maxillarygingivae. At Day 44, however, Group A receives adoptive transfer ofAa/Pp cross-reactive Th1 cell transfer in saline (i.v.), as this hasbeen shown to cause periodontal bone loss. Such Th1 cell transferconstitutes a secondary (recurrent) activation of PPAIR. Group B micereceive control saline (i.v.) injections. Animals are sacrificed onDay-51 (=21 days after injection of gels and 7 days after Th1 celltransfer). Control, non-treated C57BL/6 mice sacrificed on Day-30provide the base-line information about inflammatory response and levelof bone loss without treatment with GM-CSF/TSLP-gel. The analysisinvolves: (1) Fluorescent immunohistochemistry for the detection ofFOXP3+EGFP+ Treg cells and other inflammatory cell types, measurement ofgingival tissue cytokines and chemical mediators, and measurement ofFOXP3+EGFP+ Treg cells and other lymphocyte phenotypes in cervical lymphnodes by flow cytometry; (2) Analyses of TRAP+ osteoclasts,Periostin+/ALP+ osteoblasts and Periostin+/ALP+ ligament fibroblasts indecalcified periodontal tissues; and (3) periodontal bone lossmeasurement.

Relation Between tDCs and iTregs.

A series of studies addressed the relationship betweenGM-CSF/TSLP-induced tolerogenic DC (tDCs) and local development of Tregcells. The functional roles of chemokines and common γchain(γc)-receptor-dependent cytokines produced by GM-CSF/TSLP-induced tDCson the extra-thymic development of Treg cells. Treg cells migrate tofungus-infected lesions in a CCR5 dependent manner in a mouse model ofpulmonary mycosis, and Treg cells migrate to the infectious lesion inresponse to the CCR5-ligands, such as MIP-1α, which are also known to beexpressed by GM-CSF-stimulated DC CD25+CD4+ Treg cells can be developedby ex vivo stimulation with TGFβ a and IL-2 from whole spleen cells.Results (FIG. 8) demonstrated that ex vivo stimulation of mouse wholespleen cells with TGF-β and IL-2 up-regulated the development of FOXP3+T-cells, indicating that FOXP3+ Treg cells are expandable ex vivo inresponse to appropriate stimulation. Common γchain(γc)-receptor-dependent cytokines are required for Treg cell expansion,which is demonstrated by the lack of Treg cells in γc-gene knockoutmice. Several γc-receptor-dependent cytokines, e.g. IL-2, IL-7 andIL-15, up-regulate Treg development. Because TSLP, which also uses theγc-receptor, does not induce development of Treg cells TSLP releasedfrom the gels does not directly induce Treg development. However, DCs donot produce the major γc-receptor-dependent cytokine IL-2. Therefore,IL-15 that is produced by DC following stimulation with GM-CSF (Ge etall, 2002), facilitates Treg growth as a γc-receptor-dependent cytokine.If tDCs do not induce local development of FOXP3+ Treg cells from nTreg,then non-Treg cells, i.e., FOXP3(−)CD4(+) T cells, may migrate to the PDlesion and differentiate to FOXP3(+) iTreg cells by communication withthe tDCs. Thus, these experiments examined in vitro chemokines andcommon γchain (γc)-receptor-dependent cytokines produced byGM-CSF/TSLP-induced tDCs and their functional roles in thechemo-attraction and development of FOXP3+ Treg cells.

Measurement of cytokines and chemokines produced by GM-CSF/TSLP-inducedtDCs CD11+ DC are induced in vitro by the incubation of bone marrowcells with GM-CSF (10 ng/ml) in the presence or absence of TSLP (10ng/ml). After 7 days of incubation, CD11c+DC are isolated from the bonemarrow cell culture, using anti-CD11c MAb-conjugated MACS beads (DCisolation kit, Miltenyi Biotech). CD11c+DC are be separated frommononuclear cells (MNC) freshly isolated from the dorsal s.c. tissue ofmice where GM-CSF-gel, GM-CSF/TSLP-gel or control empty gel (GM-CSF andTSLP, 1 ug and 1 ug, respectively; 1.5 ul-gel/site) is injected 7 daysprior to the MNC isolation, using anti-CD11c MAb-conjugated MACS beads.Doses and concentrations are adjusted as necessary. These DC areincubated in vitro in the presence or absence of bacterial stimulation(fixed Aa, fixed P. gingivalis, Aa-LPS or Pg-LPS) or proinflammatoryfactor (IL1-α), and their expression level of chemokines and cytokinesis measured quantitatively by Mouse Cytokine/Chemokine Panel-24-Plex(Millipore; see Table 1) using a Luminex multiplex system. Theproduction of inflammatory chemical mediators (PGE₂, NO, ATP, andadenosine) are also monitored, although detection of ATP and adenosinefrom DCs.

In vitro assays examined the Treg cell chemo-attractant factors secretedfrom DC. The culture supernatants of Aa- or IL-1α-stimulated CD11c+DC,are placed in the bottom compartment of a transmigration system, whileFOXP3(+)EGFP(+) Treg cells, or control FOXP3(−) CD4 T-cells, are freshlyisolated from FOXP3-EGFP-KI mice by cell-sorting and applied to acell-culture insert (5 μm pore size, Millipore). The kinetics and numberof migrating FOXP3(+) Treg cells, or control FOXP3(−) CD4 T-cells, tothe bottom compartment are monitored. In order to evaluate thefunctional role of Treg attracting factors, neutralizing mAb to thechemokines is applied to the bottom compartment with the supernatant ofDC culture. MIP-1α is a Treg chemo-attractant secreted from tDCs.Recombinant chemokines serve as positive control chemo-attractantfactors in this Treg cell migration assay. The expression of CCR2, CCR5and other chemokine receptors expressed on the migrating FOXP3(+) Tregcells or control FOXP3(−) CD4 T-cells is monitored using flow cytometry.

In vitro assays examined the FOXP3+ Treg development factors secretedfrom DCs. The CD11c+DC were co-cultured with FOXP3(+)Treg cells andFOXP3(−) CD4 T-cells isolated from the spleens of FOXP3-EGFP-KI mice inthe presence or absence of Aa-antigen. After 3, 7 and 14 days ofincubation, the proportion of FOXP3(+)Treg cells are analyzed using flowcytometry. As can be observed from the scheme of possible results shownin FIG. 13, the advantage of using FOXP3-EGFP-KI mice with this assaysystem derives whether DC-mediated Treg development occurs fromFOXP3(+)Treg cells or FOXP3(−) CD4 T-cells because: (1) liveFOXP3(+)Treg cells can be isolated from FOXP3-EGFP-KI mice; and (2)development of mature Treg cells from their precursors, which do notexpress the FOXP3 gene, can be monitored by the detection of EGFPexpression. In order to evaluate the functional role of Treg growthcytokines, neutralizing mAb to the cytokines are applied to theco-culture between DC and T-cells. IL-15 may be the major Treg growthcytokine secreted from tDCs.

Inflammation is suppressed in the PD lesion by 7 days (Day-37) after theinjection of GM-CSF/TSLP-gel and that suppression effects lasts untilDay-58, the latest examination day.

Combining Anti-Inflammatory and Osteoinductive Signaling for BoneRegeneration

The utility of the immune programming system developed and studied isevaluated for its ability to enhance bone regeneration via co-deliveryof osteoinductive cues. This approach both stops inflammation andactively promotes bone regeneration via delivery of pDNA encoding forBMP-2, using the same gel that releases GM-CSF/TSLP. The utility of thegel system is enhanced by its ability to release the pDNA on demand withan external signal (ultrasound irradiation). Ultrasound provides anumber of advantages for this application, including its non-invasivenature, deep tissue penetration, and ability to be focused andcontrolled. The delivery system is used to first quench inflammation,and subsequently release pDNA to promote alveolar bone regeneration.

The first studies characterize ultrasound-triggered pDNA release fromalginate gels, and subsequent studies examine bone regeneration usingpDNA release from the gels in the PD model. Ultrasound can be used totrigger the release of pDNA from alginate gels after multiple days ofincubation. Both PEI-condensed pDNA and uncondensed pDNA areencapsulated into alginate gels, and the passive pDNA releasequantified. PEI-condensed pDNA is examined, as condensation dramaticallyupregulates pDNA uptake and expression, and the impact of ultrasound onrelease may be distinct for the two pDNA forms due to their differentsizes and charges. Gels that vary in degradation times from 2-3 weeks toover 6 months are used for pDNA encapsulation, and little to no passivepDNA release occurs in the absence of gel degradation. The influence ofvarying regimes of low-frequency ultrasound irradiation (frequency of20-50 kHz, intensity of 0.1-10 watt, duration 1-15 min) on pDNA releaseis examined after gels have incubated for times ranging from 1-3 weeks(to mimic the intended application in which GM-CSF/TSLP release occursearly and only following amelioration of inflammation will release ofpDNA encoding BMP be triggered). The concentration of DNA in the releasemedium is assayed using Hoechst 33258 dye and a fluorometer (Hoefer DyNAQuant 200, Pharmacia Biotech, Uppsala, Sweden). The structural integrityof the released plasmid is examined using gel electrophoresis. Littleeffect of ultrasound on the GM-CSF and TSLP release is anticipated, asultrasound is not initiated until after the majority of GM-CSF and TSLPhave been released, but GM-CSF and TSLP release is be monitored duringirradiation to determine if ultrasound impacts the release of anyresidual GM-CSF/TSLP remaining in the gels.

The ability of on-demand pDNA release from gels to enable in vivotransfection is examined to confirm both that ultrasound can regulatepDNA in vivo in a similar fashion as noted in vitro, and to determinethe appropriate pDNA dose for bone regeneration studies. Gels containingpDNA encoding GFP are injected into palatal maxillary gingivae of normalmice (no periodontal disease), and subjected to ultrasound at timesranging from 7-21 days after introduction. The in vitro studies are usedas a guide for the relevant frequency, intensity, and duration ofirradiation. An exemplary ultrasound schedule comprises application onceper day, for time-frames ranging from 1-7 days. One day following theend of each irradiation period, animals are sacrificed, and tissuesections obtained for both histology and biochemical quantification ofoverall GFP expression in the tissue. Uncondensed and PEI-condensed pDNAare compared in these studies, and the doses of encapsulated pDNA variedfrom 1 μg-100 μg. Tissue sections are immunostained for GFP toqualitatively study pDNA expression, and GFP levels also quantified intissue lysates to quantify expression.

Another embodiment of this invention provides for the impact of the gelsystem to first ameliorate inflammation, and then actively promoteregeneration in the PD mouse model. PD is characterized by chronicinflammation that leads to tissue destruction and bone resorption aroundthe teeth. After induction of PD, gels containing GM-CSF, TSLP, and pDNAencoding BMP-2 are injected at Day-30. After sufficient time has elapsedto allow inflammation to reside, ultrasound irradiation is initiated torelease pDNA encoding BMP-2. At 2, 4 and 8 weeks following gelplacement, the soft and hard tissue is retrieved and analyzed. The levelof inflammation is monitored by measurement of inflammatory chemicalmediators present in the gingival tissue, and BMP-2 levels is alsoquantified with ELISA to examine gene expression. Bone regeneration isquantified using micro-CT and histologic analysis is also performed toallow quantitative histomorphometry of bone quantity. Controls includeno treatment, gels containing pDNA only (no GM-CSF/TSLP), and blankgels. A sample size of 6/time point/condition is anticipated to benecessary studies of bone regeneration.

Reducing inflammation dramatically increases bone regeneration resultingfrom osteoinductive factor delivery, as compared to osteoinductivefactor alone. Ultrasound provides a useful trigger to control therelease of pDNA from alginate gels, both in vitro and in vivo, allowinga single gel to deliver the GM-CSF/TSLP and the plasmid with appropriaterelease kinetics. In some cases, there is an interplay between the geldegradation rate and ultrasound-triggered release due to the changes ingel structure resulting from degradation. Two gel injections—the firstdelivering GM-CSF/TLSP to ameliorate inflammation, and the second todelivery pDNA encoding BMP-2 after inflammation has been reduced, may beused.

High, local levels of BMP-2 significantly enhance bone regeneration. Themajor effect of ultrasound on regeneration is triggered release of pDNAfrom gels, but ultrasound also enhances cellular uptake of pDNA and thusdirectly enhances expression of locally delivered pDNA in addition orwithout effects on pDNA release.

The following materials and methods were used in periodontal studiesdescribed herein.

In Vitro DC Assays

Migration assays are performed with 6.5 mm transwell dishes (Costar,Cambridge, Mass.) with a pore size of 5 μm. The effects of GM-CSF andTSLP, (Invivogen, San Diego, Calif.) on the migration of DCs areassessed by placing recombinant murine GM-CSF and TSLP in the bottomwells and 5×10⁵ DCs in the top wells. To assess the effects of GM-CSFand TSLP on DC activation, cells are cultured with bacterial stimulation(fixed Aa, fixed P. gingivalis, Aa-LPS or Pg-LPS along) with variousconcentrations of TSLP and GM-CSF for 24 hours and then the cells arewashed and fixed in 10% formalin. The cells are prepared forfluorescence immunohistochemistry as per below, and examined usingfluorescent microscopy (Olympus, Center Valley, Pa.). Cells are alsoanalyzed by FACS, and gated according to positive stains using isotypecontrols, and the percentage of cells staining positive for each surfaceantigen will be recorded. The expression of cytokines upregulated as aresult of DC maturation is quantified as described below.

Gel Fabrication

Gels are created from alginates varying in mannuronic to guluronic acidresidues, molecular weight distributions, and extent of oxidation toregulate their rheological, physical and degradation properties.Hydrogels are prepared by mixing alginate solutions containing thefactors as previously described for proteins and plasmid DNAformulations with a calcium sulfate slurry. If necessary, factors arefirst encapsulated into PLG microspheres using a standard doubleemulsion technique.

Quantification of GM-CSF, TSLP, and pDNA In Vitro Release Studies, andIn Vivo Concentrations

To determine the efficiency of GM-CSF, TSLP, and pDNA incorporation andthe kinetics of release, ¹²⁵I-labeled factors (Perkin Elmer) areutilized as a tracer, and gels and placed in Phosphate Buffer Solution(PBS) (37° C.). At various time points, the PBS release media iscollected and amount of ¹²⁵I-factor released from the scaffolds isdetermined at each time point using a gamma counter and normalizing tothe total ¹²⁵I-factor incorporated into the gels. To assess theretention of GM-CSF bioactivity, loaded gels are placed in the top wellsof 6.5 mm transwell dishes (Costar, Cambridge, Mass.) with a pore sizeof 3 μm and the proliferation of JAWS II cells (DC cell line) culturedin the bottom wells is evaluated at various time points using cellcounts from a hemacytometer. To determine GM-CSF and TSLP concentrationsin vivo, tissue surrounding gels is excised and digested with tissueprotein extraction reagent (Pierce). After centrifugation, theconcentration of GM-CSF and TSLP in the supernatant is then analyzedwith ELISA (R&D systems), according to the manufacturers instructions.

In Vivo DC Migration and Activation Assays

Gels with various combinations of factors are injected into gingival ofmice. For histological examination gels and surrounding tissue areexcised and fixed in Z-fix solution, embedded in paraffin, and stainedwith hematoxylin and eosin. To analyze DC recruitment, gels andsurrounding tissue are excised at various time-points and the tissuedigested into single cell suspensions using a collagenase solution(Worthingtion, 250 U/ml) that was agitated at 37° C. for 45 min. Thecell suspensions are then poured through a 40 mm cell strainer toisolate cells from gel particles and the cells are pelleted and washedwith cold PBS and counted using a Z2 coulter counter (Beckman Coulter).The resultant cell populations are then stained with primary antibodiesconjugated to fluorescent markers to allow for analysis by flowcytometry. Cells are gated according to positive labels using isotypecontrols, and the percentage of cells staining positive for each surfaceantigen is recorded.

Fluorescent Immunohistochemistry

To evaluate the tissue localization pattern of specific cells ingingival tissues and cervical LN, confocal microscopic analysis isemployed. Using the 3-color staining procedure, key subsets, tDCs (cellspositive for CD11c and CD86 and IL-10), mature DCs (positive for CCR7,B7-2, MHCII), FOXP3+ T cells (EGFP, IL-10 and TGF-b), FOXP3+CD25+ Tcells (EGFP, CD25, IL-10), RANKL+CD3+ T cells (RANKL, CD3 and TNF-a) andRANKL+CD19+ B cells are stained. Expression of CD26, CD39 and CD73 onFOXP3+ T cells as well as on RANKL+CD3+ T cells, DC(CD11c+), B cells(CD19+), macrophages (F4/80+) and neutrophils (CD64+) are alsomonitored. Detection of RANKL is conducted by a combination ofbiotin-conjugated-OPG-Fc/TR-avidin. Other molecules are stained using aconventional method with primary specific-monoclonal antibody followedby secondary antibody conjugated with fluorescent dye: 1st color, FITC(emission/excitation, 488/515 nm); 2nd color, Texas Red (595/615); and3rd color, APC/Cy5.5 (595/690).

Flow Cytometry

The prevalence of various cells in gingival tissue and local cervicallymph nodes is analyzed by flow cytometry. Nonspecific antibody bindingto the Fc receptor is blocked by pre-incubating the cells with rat MAb2.4G2 (reactive to CD16/CD32). Three-color staining method is employedfor the detection of tDCs, mature DC, EGFP+FOXP3+ T cells and RANKL+CD3+T cells.

Detection of Cytokines from Culture Medium and Gingival TissueHomogenates

Standard methods were used to detect cytokines and other markers such asIL-10, RANKL, OPG, Osteocalcin, TNF-a, IFN-g, TGF-b1, IL-1b, IL-2, IL-4,IL-6, IL-12 and IL-17 in the culture medium or mouse gingival tissuehomogenates.

Detection of Inflammatory Chemical Mediators Present in Gingival Tissue

Both pro-inflammatory (PGE₂, nitric oxide [NO] and ATP) andanti-inflammatory chemical mediators (adenosine) are measured. PGE₂ ismeasured using a Luminex-based PGE₂ detection kit (Cayman Chemical).Nitric oxide present in tissue homogenate is measured by Nitrate/NitriteColorimetric Assay Kit (Cayman Chemical). The concentration of ATP andadenosine will be measured using Sarissaprobe®-ATP and Sarissaprobe®-ADOsensors (Sarissa Biomedical, Coventry, UK).

TRAP Staining for Osteoclasts and Periostin/ALP Staining for Osteoblastsand Periodontal Ligament Fibroblasts in Periodontal Bone

The maxillary jaws of animals sacrificed on Day-33, -37, -44, and -58are decalcified, and osteoclast cells determined by TRAP staining on thetissue sections. The tissue sections are also stained for Periostin andalkaline phosphatase to determine the localization of osteoblasts andperiodontal ligament fibroblasts.

pDNA Studies

Plasmid DNA containing the CMV promoter and encoding for greenfluorescent protein (GFP) (Aldevron, Fargo N. Dak.) or bonemorphogenetic protein 2 (BMP-2) (Aldevron) are used. Branchedpolyethylenimine (PEI, MW=25000, Sigma-Aldrich) is used to condenseplasmid DNA for more efficient transfection.

Application of Ultrasound

An Omnisound 3000 will be to mediate pDNA release from gels. Thestructure of gels subject to sonication in vitro are examined viaanalysis of rheological properties at varying times post-treatment todetermine permanent changes in gel structure, and recovery timepost-treatment. pDNA release, structure, and gene expression areevaluated using standard methods. For in vivo studies, a 1-cm²transducer head is used with aquasonic coupling gel on the tissuesurface; a thermocouple is inserted into the tissue site to measurelocal temperature.

Monitoring Extent of Bone Regeneration

Tissues are analyzed initially by microCT and then histologically todetermine the extent of bone formation. Digital μCT images are taken andreconstructed into a 3-dimensional image with a mesh size of 25 μm×25μm×25 μm. Scanning may be performed on a GE-EVS high resolution MicroCTSystem available at the Brigham and Woman core facility, on a per feebasis. Bone volume measures, and calibrated bone mineral density aredetermined. Quantitative histomorphometric analysis is carried out usingstandard methods, from plastic embedded sections stained with Goldner'sTrichrome stain for osteoid or von Kossa stain for mineralized tissue.

Statistical Design and Analysis

Sample numbers for all experiments are calculated using InStat Software(Agoura Hills, Calif.), using standard deviations determined inpreliminary studies, in order to enable the statistical significance ofdifferences between experimental conditions of greater than 50% to beestablished. Statistical analysis will be performed using Studentst-test (two-tail comparisons), and analyzed using InStat 2.01 software.Differences between conditions are considered significant if p<0.05.

All United States patents and published or unpublished United Statespatent applications cited herein are incorporated by reference. Allpublished foreign patents and patent applications cited herein arehereby incorporated by reference. All other published references,documents, manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

We claim:
 1. A method of reducing the severity of an autoimmunedisorder, comprising identifying a subject suffering from an autoimmunedisorder and administering to said subject a scaffold compositioncomprising an antigen, a recruitment composition, and a tolerogen,wherein said antigen is derived from a cell to which a pathologicautoimmune response associated with said disorder is directed; whereinsaid tolerogen induces immune tolerance or a reduction in an immuneresponse; wherein said tolerogen is selected from the group consistingof retinoic acid, rapamycin, aspirin, and vasoactive intestinal peptide;and wherein the scaffold composition does not comprise IL-10,dexamethasone, vitamin D, or TGF-beta.
 2. The method of claim 1, whereinsaid autoimmune disorder is type 1 diabetes.
 3. The method of claim 1,wherein said antigen comprises a pancreatic cell antigen.
 4. The methodof claim 3, wherein said antigen comprises insulin, proinsulin, glutamicacid decarboxylase-65 (GAD65), insulinoma-associated protein 2, heatshock protein 60, ZnT8, or islet-specific glucose-6-phosphatasecatalytic subunit.
 5. The method of claim 1, wherein said autoimmunedisorder is multiple sclerosis.
 6. The method of claim 5, wherein saidantigen comprises myelin basic protein, myelin proteolipid protein,myelin-associated oligodendrocyte basic protein, or myelinoligodendrocyte glycoprotein.
 7. The method of claim 1, wherein saidrecruitment composition comprises GM-CSF, FMS-like tyrosine kinase 3ligand, N-formyl peptides, fractalkine, or monocyte chemotacticprotein-1.
 8. The method of claim 7, wherein said recruitmentcomposition comprises GM-CSF.
 9. The method of claim 1, wherein saidscaffold composition comprises a non-inflammatory polymer.
 10. Themethod of claim 9, wherein said non-inflammatory polymer comprisesalginate, poly(ethylene glycol), hyaluronic acid, collagen, gelatin,poly(vinyl alcohol), fibrin, poly(glutamic acid), peptide amphiphiles,silk, fibronectin, chitin, poly(methyl methacrylate), poly(ethyleneterephthalate), poly(dimethylsiloxane), poly(tetrafluoroethylene),polyethylene, polyurethane, poly(glycolic acid), poly(lactic acid),poly(caprolactone), poly(lactide-co-glycolide) (PLGA), polydioxanone,polyglyconate, BAK; poly(ortho ester I), poly(ortho ester) II,poly(ortho ester) III, poly(ortho ester) IV, polypropylene fumarate,poly[(carboxy phenoxy)propane-sebacic acid],poly[pyromellitylimidoalanine-co-1,6-bis(p-carboxy phenoxy)hexane],polyphosphazene, starch, cellulose, albumin, polyhydroxyalkanoates,poly(lactide), or poly(glycolide).
 11. The method of claim 1, whereinsaid scaffold composition comprises a hydrogel.
 12. The method of claim11, wherein said hydrogel comprises an alginate gel polymer.
 13. Themethod of claim 12, wherein said hydrogel comprises 1-5% alginate gelpolymer.
 14. The method of claim 13, wherein said alginate gel polymeris crosslinked.
 15. The method of claim 1, wherein the tolerogen isencapsulated in poly(lactide-co-glycolide) (PLGA) microspheres.
 16. Themethod of claim 1, wherein said scaffold composition is administered byinjection, implantation, topically affixing a skin patch comprising thescaffold composition, or delivering the scaffold composition by aerosolinto a lung or nasal passage of the subject.
 17. The method of claim 16,wherein said scaffold composition is administered by intradermalimplantation.
 18. The method of claim 1, wherein said autoimmunedisorder is Crohn's disease, rheumatoid arthritis, Systemic lupuserythematosus, Scleroderma, Alopecia areata, Antiphospholipid antibodysyndrome, Autoimmune hepatitis, Celiac disease, Graves' disease,Guillain-Barre syndrome, Hashimoto's disease, Hemolytic anemia,Idiopathic thrombocytopenic purpura, inflammatory bowel disease,ulcerative colitis, inflammatory myopathies, Polymyositis, Myastheniagravis, Primary biliary cirrhosis, Psoriasis, Sjogren's syndrome,Vitiligo, gout, celiac disease, atopic dermatitis, acne vulgaris,autoimmune hepatitis, or autoimmune pancreatitis.
 19. The method ofclaim 1, wherein the scaffold comprises 0.1 μg to 10 μg of saidtolerogen.
 20. The method of claim 1, wherein the scaffold comprises 0.1μg to 10 μg of said recruitment composition.
 21. The method of claim 1,wherein said scaffold composition comprises an alginate gel andgranulocyte macrophage colony-stimulating factor (GM-CSF).